Plants tolerant to HPPD inhibitor herbicides

ABSTRACT

The present invention relates to nucleic acid sequences encoding a hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, abbreviated herein as HPPD) obtained from Euryarchaeota belonging to the family Picrophilaceae, as well as the proteins encoded thereby, and to a chimeric gene which comprises such nucleic acid sequence, and to the use of such nucleic acid sequences, proteins or chimeric genes for obtaining plants which are tolerant to HPPD inhibitor herbicides.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (name:2400_(—)3880001_sequencelisting_ascii.txt (58 KB); date of creation:Mar. 4, 2011) submitted in this application is incorporated herein byreference in its entirety.

The present invention relates to nucleic acid sequences encoding ahydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, abbreviated herein asHPPD) obtained from Euryarchaeota belonging to the familyPicrophilaceae, as well as the proteins encoded thereby, and to achimeric gene which comprises such nucleic acid sequence, and to the useof such nucleic acid sequences, proteins or chimeric genes for obtainingplants which are tolerant to HPPD inhibitor herbicides.

BACKGROUND

The HPPDs are enzymes which catalyse the reaction in whichpara-hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosinedegradation product, is transformed into homogentisate (abbreviatedherein as HG), the precursor in plants of tocopherol and plastoquinone(Crouch N. P. et al. (1997) Tetrahedron, 53, 20, 6993-7010, Fritze etal., (2004), Plant Physiology 134:1388-1400). Tocopherol acts as amembrane-associated antioxidant. Plastoquinone, firstly acts as anelectron carrier between PSII and the cytochrome b6/f complex andsecondly, is a redox cofactor for phytoene desaturase, which is involvedin the biosynthesis of carotenoids.

Up to now, more than 700 nucleic acid sequences from various organismspresent in NCBI database were annotated as coding for a putative proteinhaving an HPPD domain including the sequence disclosed under the Q6KZ98accession number given in the UniProtKB/TrEMBL database as well as theYP_(—)024147 accession number given in the NCBI protein database. Butfor most of those, including the sequence corresponding to the accessionnumber Q6KZ98/YP_(—)024147, it has not been proven that the proteinwould have an HPPD enzymatic activity either in an in vitro assay or anin in planta approach, nor that such HPPD protein can confer herbicidetolerance to HPPD inhibitor herbicides when expressed in a plant.Several HPPD proteins and their primary sequences have been described inthe state of the art, in particular the HPPD proteins of bacteria suchas Pseudomonas (Rüetschi et al., Eur. J. Biochem., 205, 459-466, 1992,WO 96/38567), of plants such as Arabidopsis (WO 96/38567, GenebankAF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa (WO02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO 02/046387),Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO 02/046387),Festuca arundinacea (WO 02/046387), Setaria faberi (WO 02/046387),Eleusine indica (WO 02/046387), Sorghum (WO 02/046387), Coccicoides(Genebank COITRP), of Coptis japonica (WO 06/132270), Chlamydomonasreinhardtii (ES 2275365), or of mammals such as mouse or pig. Thecorresponding sequences disclosed in the indicated references are herebyincorporated by reference.

Most plants synthesize tyrosine via arrogenate (Abou-Zeid et al. (1995),Applied Env Microb 41: 1298-1302; Bonner et al., (1995), Plant CellsPhysiol. 36, 1013-1022; Byng et al., (1981), Phytochemistry 6:1289-1292; Connely and Conn (1986), Z. Naturforsch 41c: 69-78; Gaines etal., (1982), Plants 156: 233-240). In these plants, the HPP is derivedonly from the degradation of tyrosine. On the other hand, in organismssuch as the yeast Saccharomyces cerevisiae or the bacterium Escherichiacoli, HPP is a tyrosine precursor, and it is synthesized by the actionof an enzyme, prephenate dehydrogenase (hereinafter referred to as PDH),which converts prephenate to HPP (Lingens et al., (1967) European J.Biochem 1: 363-374; Sampathkumar and Morrisson (1982), Bioch BiophysActa 701: 204-211). In these organisms, the production of HPP istherefore directly connected to the aromatic amino acid biosyntheticpathway (shikimate pathway), and not to the tyrosine degradationpathway.

Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency inaccessory light-harvesting pigments and, most importantly, todestruction of chlorophyll by UV-radiation and reactive oxygen species(bleaching) due to the lack of photo protection normally provided bycarotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149). Bleachingof photosynthetically active tissues leads to growth inhibition andplant death.

Some molecules which inhibit HPPD, and which bind specifically to theenzyme in order to inhibit transformation of the HPP into homogentisate,have proven to be very effective selective herbicides.

At present, most commercially available HPPD inhibitor herbicides belongto one of these four chemical families:

1) the triketones, e.g. sulcotrione [i.e.2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione], mesotrione[i.e. 2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione];tembotrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2,-tri-fluoroethoxy)methyl]benzoyl]-1,3-cyclo-hexanedione];tefuryltrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoyl]-1,3-cyclohexanedione]];bicyclopyrone [i.e.4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one];Benzobicyclon [i.e.3-(2-chloro-4-mesylbenzoyl)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one]

2) the diketonitriles, e.g.2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione;

3) the isoxazoles, e.g. isoxaflutole [i.e.(5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone].In plants, the isoxaflutole is rapidly metabolized in DKN, adiketonitrile compound which exhibits the HPPD inhibitor property; and

4) the pyrazolinates, e.g. topramezone[i.e.[3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone],and pyrasulfotole[(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluaromethylphenyl)methanone];pyrazofen[2-[4-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].

These HPPD-inhibiting herbicides can be used against grass and/or broadleaf weeds in crop plants that display metabolic tolerance, such asmaize (Zea mays) in which they are rapidly degraded (Schulz et al.,(1993). FEBS letters, 318, 162-166; Mitchell et al., (2001) PestManagement Science, Vol 57, 120-128; Garcia et al., (2000) Biochem., 39,7501-7507; Pallett et al., (2001) Pest Management Science, Vol 57,133-142). In order to extend the scope of these HPPD-inhibitingherbicides, several efforts have been developed in order to confer toplants, particularly plants without or with an underperforming metabolictolerance, a tolerance level acceptable under agronomic fieldconditions.

Besides the attempt of by-passing HPPD-mediated production ofhomogentisate (U.S. Pat. No. 6,812,010), overexpressing the sensitiveenzyme so as to produce quantities of the target enzyme in the plantwhich are sufficient in relation to the herbicide has been performed(WO96/38567). Overexpression of HPPD resulted in better pre-emergencetolerance to the diketonitrile derivative (DKN) of isoxaflutole (IFT),but tolerance was not sufficient for tolerance to post-emergencetreatment (Matringe et al., (2005), Pest Management Science 61:269-276).

A third strategy was to mutate the HPPD in order to obtain a targetenzyme which, while retaining its properties of catalysing thetransformation of HPP into homogentisate, is less sensitive to HPPDinhibitors than is the native HPPD before mutation.

This strategy has been successfully applied for the production of plantstolerant to2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand to2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione(EP496630), two HPPD-inhibiting herbicides belonging to thediketonitriles family (WO 99/24585). Pro215Leu, Gly336Glu, Gly336Ile,and more particularly Gly336Trp (positions of the mutated amino acid areindicated with reference to the Pseudomonas HPPD) were identified asmutations which are responsible for an increased tolerance topre-emergence treatment with these diketonitrile herbicides withoutcausing an alteration of the activity of the enzyme.

More recently, introduction of a Pseudomonas HPPD gene into the plastidgenome of tobacco and soybean has shown to be more effective thannuclear transformation, conferring even tolerance to post-emergenceapplication of isoxaflutole (Dufourmantel et al., 2007, Plant BiotechnolJ. 5(1):118-33).

In WO 04/024928, the inventors have sought to increase the prenylquinonebiosynthesis (e.g., synthesis of plastoquinones, tocopherols) in thecells of plants by increasing the flux of the HPP precursor into thecells of these plants. This has been done by connecting the synthesis ofsaid precursor to the “shikimate” pathway by overexpression of a PDHenzyme. They have also noted that the transformation of plants with agene encoding a PDH enzyme makes it possible to increase the toleranceof said plants to HPPD inhibitors.

In the patent application WO 2009/144079, a nucleic acid sequenceencoding a mutated hydroxyphenylpyruvate dioxygenase (HPPD) at position336 of the Pseudomonas fluorescens HPPD protein and its use forobtaining plants which are tolerant to HPPD inhibitor herbicides isdisclosed.

In WO 2002/046387, several domains of HPPD proteins originating fromplants have been identified that may be relevant to confer tolerance tovarious HPPD inhibitor herbicides but no in planta nor biochemical datahave been shown to confirm the impact of the as described domainfunctions.

In WO 2008/150473, the combination of two distinct tolerancemechanisms—a modified Avena sativa gene coding for a mutant HPPD enzymeand a CYP450 Maize monooxygenase (nsf1 gene)—was exemplified in order toobtain an improved tolerance to HPPD inhibitor herbicides, but no datahave been disclosed demonstrating the synergistic effects based on thecombination of both proteins.

Despite these successes obtained for the development of plants showingtolerance to several HPPD inhibitors herbicides described above, it isstill necessary to develop and/or improve the tolerance of plants tonewer or to several different HPPD inhibitors, particularly HPPDinhibitors belonging to the classes of the triketones (e.g.sulcotrione,mesotrione, tembotrione, benzobicyclon and bicyclopyrone) and thepyrazolinates (e.g., topramezone and pyrasulfotole).

DESCRIPTION

The present invention therefore relates to the generation of transgenicplants containing a gene encoding an HPPD protein obtainable or obtainedfrom an organism belonging to the family of Picrophilaceae, and variantsor mutants thereof, more especially to a gene from an organism belongingto the genus Picrophilus, and variants or mutants thereof, coding for anHPPD enzyme showing the properties of catalysing the conversion ofpara-hydroxyphenylpyruvate to homogentisate and which plants are lesssensitive to HPPD inhibitors than plants not containing any such HPPDencoding transgene.

More especially, the present invention therefore relates to thegeneration of transgenic plants containing a gene obtainable or obtainedfrom an organism belonging to the family of Picrophilaceae, especiallyfrom the genus Picrophilus, more especially obtained from the speciesPicrophilus torridus, and variants or mutants thereof, coding for anHPPD enzyme showing the properties of catalysing the conversion ofpara-hydroxyphenylpyruvate to homogentisate and which are less sensitiveto HPPD inhibitors than plants not containing any such HPPD transgene.The genes from Picrophilaceae, especially from the genus Picrophiluscoding for HPPD proteins were selected as excellent HPPD-inhibitortolerant candidates due to their high divergences in the amino acidscomposition at positions relevant for HPPD inhibitor tolerance asdetermined experimentally and structurally in the HPPD protein comparedto the sensitive Arabidopsis HPPD protein which was taken as theHPPD-inhibitor herbicide sensitive reference molecule.

In one embodiment, this invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particularly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6 or 7, preferably SEQ IDNo. 6.

In a further embodiment, the invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particurlarly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6, 7, preferably SEQ IDNo. 6, and in which any amino acids from position 177 to position 368 ofSEQ ID No. 4 can be amended by any naturally-occurring amino acid,preferentially it can be any conservative substitution.

In a further embodiment, the invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particurlarly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6, 7, preferably SEQ IDNo. 6, and having one or more of the following amino acids at theposition defined by its number (relating to the number of SEQ ID No. 4)given in brackets, i.e. His(175), Ser(218), Asn(232), Gln(256),His(257), Tyr(286), Gln(321), Phe(334), Glu(336), Gly(347), andAsn(350).

In a further embodiment, the invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particularly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6, 7, preferably SEQ IDNo. 6, and at the respective positions given in the second column ofTable (i) the originally occurring amino acids can substituted by any ofthe amino acids listed in column 3 of Table (i).

TABLE (i) Amino acid in Position SEQ ID in SEQ No. 4 ID No. 4Substitutions Val 177 Thr, Cys, Ala, Gly Phe, Tyr, Ile, Val, Ala, Gln,Glu, Asp, Gly, Thr, Ser, Met, Arg, Leu 201 Lys Ile 202 Ala, Trp, Leu,Ser, Arg, Lys, His, Asp, Glu, Pro, Gly, Asn Phe 204 Val, Ile, Ala, Leu,Trp, Met, Gln, His Leu 216 Met, Val Lys 219 Ala, Val, Leu, Met, Ile,Arg, Gln, Tyr Val 221 Leu, Met, Ile, Ala Lys 222 Ala, Ser, Thr, Val,Arg, Glu, Leu, Ile, Met, His Ala 353 Glu, Gln, Ser, Val, Phe, Thr Leu354 Arg

In a further embodiment, the invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particularly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6, 7, preferably SEQ IDNo. 6, and at the respective positions given in the second column ofTable (ii) the originally occurring amino acids can substituted by anyof the amino acids listed in column 3 of Table (ii).

TABLE (ii) Amino acid in Position SEQ ID in SEQ No. 4 ID No. 4Substitutions Thr 203 Glu, Ser, Tyr, Phe, His, Gln, Asn, Gly, Leu, Met,Val, Arg, Ile Val 220 Ala, Thr Pro 230 Ala, Val, Thr, Asn, Ile, Leu 280Met, Ile, Asn Leu 310 Met Asn 348 Any except Pro Gly 349 Ala, Pro, Val,Thr, Met

In a further embodiment, the invention relates to an HPPD protein namedherein “the HPPD protein of this invention” or “the Picrophilus HPPDprotein”, which is an HPPD protein with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%; at least 97%; at least 98%, or atleast 99% amino acid sequence identity to the amino acid sequence of SEQID No. 4 from amino acid position 2 to 368, particularly to the aminoacid sequence of any one of SEQ ID Nos. 4, 5, 6, 7, preferably SEQ IDNo. 6, and at the respective positions given in the second column ofTable (iii) the originally occurring amino acids can substituted by anyof the amino acids listed in column 3 of Table (iii).

TABLE (iii) Amino acid in Position SEQ ID in SEQ No. 4 ID No. 4Substitutions Thr 203 Glu, Ser, Arg, Tyr Val 220 Ala Pro 230 Ala, Val,Thr Leu 280 Met Leu 310 Met Asn 348 Ile, Ala, Val, Leu, Lys Gly 349 Ala

This includes a protein with amino acids substituted, deleted or addedcompared to the sequence of SEQ ID No. 4 from amino acid position 2 toamino acid position 368, such as a transit peptide fusion protein, or aprotein with amino acid changes in the sequence of SEQ ID No. 4 thatretains the enzymatic function of an HPPD protein, and that stillconfers HPPD tolerance when expressed in plants, preferably HPPDtolerance of comparable range to that conferred by the protein of SEQ IDNo. 4. This includes variant or mutant proteins derived from the proteinof SEQ ID No. 4, such as any of the proteins of SEQ ID Nos 5, 6 or 7,particularly such mutant or variant which is less sensitive than thehost plant's endogenous HPPD to an HPPD inhibitor herbicide of the classof isoxazoles, diketonitriles, triketones or pyrazolinates, preferablysuch mutant or variant which confers agronomically relevant herbicidetolerance to a host plant expressing it when an HPPD inhibitor herbicideof the class of isoxazoles, diketonitriles, triketones and/orpyrazolinates, particularly any one of mesotrione, tembotrione,isoxaflutole or bicyclopyrone is applied on such plants, moreparticularly when applied post-emergence. This also includes a proteincomprising an active portion of the sequence of SEQ ID No. 4, whichportion confers HPPD inhibitor tolerance when expressed in plants. Thisincludes a protein with substantially the same amino acid sequence asthe sequence of SEQ ID No. 4, such as a protein with the amino acidsequence of any one of SEQ ID Nos 4 to 7. This includes isolatedproteins as defined below, and also proteins, such as the protein of SEQID No. 4 wherein certain amino acids have been replaced by similar aminoacids as defined below, preferably conservative amino acidsubstitutions. Also included herein as HPPD proteins of this inventionare HPPD proteins comprising the amino acid sequence of SEQ ID No. 4from amino acid position 2 to 368, but wherein 1-20, 1-15, 1-10, or 1,2, 3, 4, 5, 6, 7, 8, or 9 amino acids have been deleted or have beensubstituted by other amino acids, particularly such protein whichretains HPPD enzymatic activity and which confers tolerance to HPPDinhibitor herbicides when expressed in a host plant. Included herein areHPPD proteins encoded by DNA sequences homologous to the DNA sequencesof the invention as described below, or HPPD proteins encoded by a DNAsequence which hybridizes to at least a portion (of at least 20-30nucleotides) of the DNA of SEQ ID No. 1, or which is obtainable using aprimer based on SEQ ID No. 1, or HPPD proteins with at least 75%sequence identity to SEQ ID No. 4 which are encoded by a DNA sequencefound in the genome sequence of a microorganism, such as a eukaryoticmicroorganism, particularly a Euryarchaeota, such as a microorganism ofthe family Picrophilus. Included herein as an HPPD protein of thisinvention is a Picrophilus HPPD protein which confers herbicidetolerance to plants when expressed in such plants, wherein suchtolerance is to an HPPD inhibitor such as mesotrione, tembotrione,isoxaflutole or bicyclopyrone, particularly such HPPD protein is aPicrophilus torridus HPPD protein, such as a protein comprising thesequence of SEQ ID No. 4 from amino acid position 2 to 368. Thisincludes the mutant or variant HPPD proteins as described further below.

The present invention includes and provides an antibody capable ofspecifically binding a substantially purified protein comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 4,5, 6, or 7, or derived sequences thereof according to amino acidreplacement as disclosed in one or more of tables (i), (ii) or (iii),above.

A further aspect of the invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the protein or peptide molecules of the invention andtheir homologs, fusions or fragments. In a particularly preferredembodiment, the antibody specifically binds to a protein having theamino acid sequence set forth in SEQ ID NOs: 4-7 or a fragment thereof,or derived sequences thereof according to amino acid replacement asdisclosed in one or more of tables (i), (ii) or (iii), above.

In another embodiment, the antibody specifically binds to a fusionprotein comprising an amino acid sequence selected from the amino acidsequence set forth in SEQ ID NOs: 4-7 or a fragment thereof, or derivedsequences thereof according to amino acid replacement as disclosed inone or more of tables (i), (ii) or (iii), above.

In another embodiment the antibody specifically binds to a fusionprotein comprising an amino acid sequence selected from the amino acidsequence set forth in SEQ ID NOs: 4-7 or a fragment thereof, or derivedsequences thereof according to amino acid replacement as disclosed inone or more of tables (i), (ii) or (iii), above.

Antibodies of the invention may be used to quantitatively orqualitatively detect the protein or peptide molecules of the invention,or to detect post translational modifications of the proteins. As usedherein, an antibody or peptide is said to “specifically bind” to aprotein or peptide molecule of the invention if such binding is notcompetitively inhibited by the presence of non-related molecules.

In another embodiment this invention relates to an HPPD nucleic acid orDNA, named herein “the HPPD nucleic acid/DNA of this invention”, whichis a nucleic acid or DNA encoding an HPPD of this invention as definedabove. This includes a DNA which comprises a nucleotide sequenceselected from the group consisting of the sequence of SEQ ID No. 1 fromnucleotide position 4 to nucleotide position 1104, the sequence of SEQID No. 2 from nucleotide position 25 to nucleotide position 1125, or thesequence of SEQ ID No. 3 from nucleotide position 4 to nucleotideposition 1500, or which comprises a DNA region which encodes an HPPD, ora DNA which is sufficiently complementary to another DNA so that when itis incubated at a temperature of between 60 and 65° C. in 5×SSC (1×SSC(single-strength sodium citrate) means=0.15M NaCl, 0.015 Mtrisodium-citrate, 50 mM sodium phosphate pH 7.6), containing 0.1% SDSfollowed by rinsing at the same temperature with 0.3 5×SSC containing0.1% SDS, it still hybridizes with a sequence selected from the groupconsisting of SEQ ID Nos. 1, 2, and 3. When the test and inventivesequences are double stranded the nucleic acid constituting the testsequence preferably has a TM within 10° C. of that of the sequenceselected from the group consisting of SEQ ID Nos 1, 2, and 3. In thecase that the test and the sequence selected from the group consistingof SEQ ID Nos. 1, 2, and 3 are mixed together and are denaturedsimultaneously, the TM values of the sequences are preferably within 5°C. of each other. More preferably the hybridization is performed underrelatively stringent hybridization conditions as defined below. In oneembodiment, a denatured test or inventive sequence is preferably firstbound to a support and hybridization is effected for a specified periodof time at a temperature of between 60 and 65° C. in 5×SSC containing0.1% SDS followed by rinsing of the support at the same temperature butwith 0.1×SSC. Where the hybridization involves a fragment of thesequence selected from the group consisting of SEQ ID Nos. 1, 2, and 3the hybridization conditions may be less stringent, as will be obviousto the skilled person.

Also included herein as HPPD DNA of this invention, are DNA sequencesencoding an HPPD protein of the invention which DNA sequences have beenadapted for expression in microorganisms or plants, such as by replacingnatice codons by codons more preferred in a host cell, or whereincertain restriction sites have been added or removed for ease ofcloning, or DNA sequence with a certain number of added, replaced ordeleted nucleotides. This also includes isolated DNA sequences andvariant, mutant or synthetic DNAs or nucleic acids as described furtherbelow.

In a particular embodiment, the Picrophilus HPPD DNA of this inventionis expressed in plants under the control of a promoter that allowsexpression of exogenous genes in plants. In a further particularembodiment, at the N-terminus of the so expressed HPPD enzyme a signalpeptide, such as a transit peptide is located, preferably a plastidtransit peptide, such as a chloroplast transit peptide of about 120amino acids (about 30 to about 120 amino acids) most preferably a doubletransit peptide, such as an optimized transit peptide of which the firstpart is originated from Sunflower (Helianthus annuus) and the secondpart from Zea mays (described in U.S. Pat. No. 5,188,642) or a plastidtransit peptide of that of the plant ribulose biscarboxylase/oxygenasesmall subunit (RuBisCO ssu), where appropriate including a few aminoacids of the N-terminal part of the mature RuBisCO ssu (EP 189 707)

In a further particular embodiment, this invention includes a DNAencoding an HPPD protein of this invention which is derived or isobtainable from SEQ ID No. 1 and is optimized for the expression in E.coli, such as a codon-optimized DNA, for example a DNA comprising thesequence of SEQ ID No. 2 from nucleotide position 25 to nucleotideposition 1125 (including the positions defined).

In a further particular embodiment, this invention includes a DNAencoding an HPPD protein of this invention which is derived from SEQ IDNo. 1 and optimized for the expression in plants, such as acodon-optimized DNA, for example a DNA comprising the sequence of “SEQID No. 3 from nucleotide position 400 to nucleotide position 1500(including the positions defined).

In a further particular embodiment, the HPPD of the invention, such asthe HPPD comprising the amino acid sequence of SEQ ID No. 4 from aminoacid position 2 to amino acid position 368, or the HPPD comprising theamino acid sequence of any one of SEQ ID Nos. 4 to 7, is less sensitivethan the host plant endogenous HPPD to an HPPD inhibitor herbicide ofthe class of isoxazoles, diketonitriles, triketones or pyrazolinates, oran HPPD inhibitor herbicide selected from isoxaflutole, tembotrione,mesotrione, sulcotrione, pyrasulfotole, topramezone,2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3 Cl₂ phenyl)propane-1,3-dione,bicyclopyrone, benzobicyclon, tefuryltrione, and pyrazoxyfen.

In a further particular embodiment, this invention includes a DNAencoding an HPPD protein of this invention which is derived from SEQ IDNo. 1 and optimized for the expression in E. coli, such as acodon-optimized DNA, for example a DNA comprising the sequence of “SEQID No. 2 from nucleotide position 25 to nucleotide position 1125(including the positions defined) which encodes an HPPD less sensitivethan the host plant endogenous HPPD to at least one HPPD inhibitorherbicide of the class of isoxazoles, diketonitriles, triketones orpyrazolinates, preferably to tembotrione, mesotrione, bicyclopyrone,tefuryltrione, isoxaflutole, diketonitrile, pyrasulfotole, topramezone,sulcotrione, pyrazolate and benzofenap.

In a further particular embodiment, this invention includes a DNAencoding an HPPD protein of this invention which is optimized for theexpression in plants, such as a codon-optimized DNA, for example a DNAcomprising the sequence of SEQ ID No. 3 from nucleotide position 400 tonucleotide position 1500 (including the positions defined) which encodesan HPPD less sensitive than the host plant endogenous HPPD to at leastone HPPD inhibitor herbicide of the class of isoxazoles, diketonitriles,triketones or pyrazolinates, preferably to tembotrione, mesotrione,bicyclopyrone, tefuryltrione, isoxaflutole, diketonitrile,pyrasulfotole, topramezone, sulcotrione, pyrazolate and benzofenap.

In a further particular embodiment, this invention relates to plants,plant parts, plant cells, and progenies of these plants comprising a DNAencoding an HPPD protein of this invention which is optimized for theexpression in E. coli, or is optimized for the expression in plants suchas a codon-optimized DNA, for example a DNA comprising the sequence ofSEQ ID No. 2 from nucleotide position 25 to nucleotide position 1125(including the positions defined) or of SEQ ID No. 3 from nucleotideposition 400 to nucleotide position 1500 (including the positionsdefined) which encodes an HPPD less sensitive than the host plantendogenous HPPD. Such plants include but are not limited to field crops,fruits and vegetables such as canola, sunflower, tobacco, sugarbeet,cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach,apple, pear, strawberry, banana, melon, potato, carrot, lettuce,cabbage, onion, soya spp, sugar cane, pea, field beans, poplar, grape,citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseedrape, and nut producing plants.

In a more particular embodiment, this invention relates to plants, plantparts, plant cells, and progenies of these plants comprising any of theDNA encoding an HPPD protein of the invention which is optimized for theexpression in E. coli, or optimized for the expression in plants such asa codon-optimized DNA, for example a DNA comprising the sequence of “SEQID No. 2 from nucleotide position 25 to nucleotide position 1125(including the positions defined) or of SEQ ID No. 3 from nucleotideposition 400 to nucleotide position 1500 (including the positionsdefined) which encodes an HPPD less sensitive than the host plantendogenous HPPD and wherein the plants are selected from the groupconsisting of canola, sunflower, tobacco, sugarbeet, cotton, maize,wheat, barley, rice, potato, soya spp, sugar cane, pea, field beans,poplar, grape, alfalfa, rye, oats, turf and forage grasses, flax andoilseed rape, and nut producing plants, even more preferably such plantsare selected from the group consisting of soya spp, rice, sugarbeet,wheat, cotton canola, oilseed rape or maize.

In another particular embodiment, the HPPD protein of the inventioncomprises the sequence of SEQ ID No. 7 and is less sensitive to an HPPDinhibitor of the class of triketones (named triketone HPPD inhibitor),such as tembotrione, sulcotrione mesotrione, bicyclopyrone,tefuryltrione, particularly tembotrione, or of the class diketonitrile(isoxaflutole) or of the class of pyrazolinates (named pyrazolinate HPPDinhibitor), such as pyrasulfotole, pyrazolate, topramezone, benzofenapcompared to the endogenous unmutated HPPD of a plant, particularly thehost plant wherein such HPPD of the invention is expressed or is to beexpressed.

The enzymatic activity of HPPD proteins can be measured by any methodthat makes it possible either to measure the decrease in the amount ofthe HPP or O₂ substrates, or to measure the accumulation of any of theproducts derived from the enzymatic reaction, i.e. homogentisate or CO₂.In particular, the HPPD activity can be measured by means of the methoddescribed in Garcia et al. (1997), Biochem. J. 325, 761-769 or Garcia etal. (1999), Plant Physiol. 119, 1507-1516, which are incorporated hereinby reference.

According to the invention, an HPPD inhibitor of the class of triketones(or triketone HPPD inhibitor) means an HPPD inhibitor having a triketoneskeleton. As an example of such triketone HPPD inhibitor, one can citethe molecules sulcotrione [i.e.2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione], mesotrione[i.e. 2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione], andtembotrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2,-tri-fluoroethoxy)methyl]benzoyl]-1,3-cyclo-hexanedione],tefuryltrione [i.e.2-{2-chloro-4-mesyl-3-[(IRS)-tetrahydro-2-furylmethoxymethyl]benzoyl}cyclohexane-1,3-dione],bicyclopyrone [i.e.4-hydroxy-3-{2-[(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridylcarbonyl}bicyclo[3.2.1]oct-3-en-2-one],benzobicyclon [i.e.3-(2-chloro-4-mesylbenzoyl)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one].

According to the invention, an HPPD of the class of pyrazolinates (orpyrazolinate HPPD inhibitor) means an HPPD inhibitor having a pyrazoleradical. As an example of such pyrazolinates HPPD inhibitor, one cancite the molecules topramezone[i.e.[3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone]and pyrasulfotole[(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4-trifluaromethylphenyl)methanone].

The present invention also relates to a nucleic acid sequence,particularly an isolated DNA, preferably a plant-expressible chimericgene, which encodes the Picrophilus HPPD of the invention and adaptedsequences thereof.

The present invention also relates to a nucleic acid sequence encodingan HPPD enzyme of this invention which retains its properties ofcatalysing the conversion of para-hydroxyphenylpyruvate to homogentisateand which is less sensitive to HPPD inhibitors of the class oftriketones such as tembotrione, sulcotrione and mesotrione, or of theclass of pyrazolinates such as pyrasulfotole and topramezone,tefuryltrione, bicyclopyrone, benzobicyclon than the endogenousunmutated plant HPPD, and of which the encoded amino acid sequence showsa sequence identity to SEQ ID No. 4 of at least 75%, 80%, particularlyat least 85%, preferably at least 90%, more preferably at least 95%,even more preferably at least 98% and most preferably at least 99%.

In a more particular embodiment, the nucleic acid sequence of theinvention encodes an HPPD enzyme which is less sensitive to an HPPDinhibitor of the class of triketones such as tembotrione, sulcotrione,mesotrione, bicyclopyrone, and tefuryltrione, the class of isoxazolessuch as isoxaflutole of the class of pyrazolinates (named pyrazolinateHPPD inhibitor), such as pyrasulfotole, pyrazolate, topramezone,benzofenap, or the class of diketones such as diketonitrile than thehost plant endogenous HPPD.

According to the present invention, a “nucleic acid sequence” isunderstood as being a nucleotide sequence which can be of the DNA or RNAtype, preferably of the DNA type, and in particular double-stranded,whether it be of natural or synthetic origin, in particular a DNAsequence in which the codons which encode the HPPD according to theinvention have been optimized in accordance with the host organism inwhich it is to be expressed (e.g., by replacing codons with those codonsmore preferred or most preferred in codon usage tables of such hostorganism or the group to which such host organism belongs, compared tothe original or source organism). An “isolated nucleicacid/DNA/protein”, as used herein, refers to a nucleic acid/DNA/proteinwhich is not naturally occurring (such as an artificial or synthetic DNAwith a different nucleotide sequence than the naturally occurring DNA,or a modified protein) or which is no longer in the natural environmentwherein it was originally present, e.g., a DNA coding sequenceassociated with a heterologous regulatory element (such as a bacterialcoding sequence operably linked to a plant-expressible promoter) in achimeric gene, a DNA transferred into another host cell, such as atransgenic plant cell.

In view of a particular embodiment of the invention and the sought-aftersolution, i.e. an HPPD which is less sensitive to a triketone, anisoxazole, or pyrazolinate HPPD inhibitor, the tolerance levelmeasurement is analyzed using the method extensively described in WO2009/14407 as described below using a triketone, an isoxazole, or apyrazolinate HPPD inhibitor, particularly an HPPD inhibitor selectedfrom tembotrione, mesotrione, pyrasulfotole, topramezone sulcotrione,bicyclopyrone, diketonitrile, benzofenap, pyrazolate, tefuryltrione.

The terminology DNA or protein “comprising” a certain sequence “X”, asused throughout the text, refers to a DNA or protein including orcontaining at least the sequence “X”, so that other nucleotide or aminoacid sequences can be included at the 5′ (or N-terminal) and/or 3′ (orC-terminal) end, e.g. (the nucleotide sequence of) a selectable markerprotein, (the nucleotide sequence of) a transit peptide, and/or a 5′leader sequence or a 3′ trailer sequence. Similarly, use of the term“comprise”, “comprising” or “comprises” throughout the text and theclaims of this application should be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

In one embodiment of the invention, the coding regions encoding HPPDcomprise a nucleotide sequence encoding proteins with the amino acidsequences as set forth in SEQ ID Nos 4, 5, 6, and 7 such as thenucleotide sequences of SEQ ID Nos 1, 2, and 3.

However, it will be clear that variants of these nucleotide sequences,including insertions, deletions and substitutions thereof may be also beused to the same effect. Equally, homologues to the mentioned nucleotidesequences from species different from Picrophilus torridus can be used.

Variants of the described nucleotide sequence will have a sequenceidentity which is preferably at least about 70%, 80%, 85% or 90% or atleast 95% with identified nucleotide sequences encoding HPPD enzymessuch as the ones identified in the sequence listing.

A protein with “substantially the same amino acid sequence” to a proteinas described in the invention, as used herein, refers to a protein withat least 90%, particularly at least 95%, preferably at least 97%sequence identity with a protein according to the invention, wherein thepercentage sequence identity is determined by using the blosum62 scoringmatrix in the GAP program of the Wisconsin package of GCG (Madison,Wis., USA) version 10.0 (GCG defaults used). “Sequence identity”, asused throughout this application, when related to proteins, refers tothe percentage of identical amino acids using this specified analysis.The “sequence identity”, as used herein, when related to DNA sequences,is determined by using the nwsgapdna scoring matrix in the GAP programof the Wisconsin package of GCG (Madison, Wis., USA) version 10.0 (GCGdefaults used).

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other, is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). The computer-assisted sequence alignment above, can beconveniently performed using standard software program such as GAP whichis part of the Wisconsin Package Version 10.1 (Genetics Computer Group,Madision, Wis., USA) using the default scoring matrix with a gapcreation penalty of 50 and a gap extension penalty of 3.

Nucleotide sequences homologous to the nucleotide sequences encoding anHPPD enzyme according to the invention may be identified by in silicoanalysis of genomic sequence data.

Homologous nucleotide sequence may also be identified and isolated byhybridization under stringent conditions using as probes identifiednucleotide sequences encoding HPPD enzymes according to the invention orparts thereof. Such parts should preferably have a nucleotide sequencecomprising at least 40 consecutive nucleotides from the coding region ofHPPD encoding genes sequences according to the invention, preferablyfrom the coding region of SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3.The probes may however comprise longer regions of nucleotide sequencesderived from the HPPD encoding nucleic acids, such as about 50, 60, 75,100, 200 or 500 consecutive nucleotides from any of the mentioned HPPDgenes. Preferably, the probe should comprise a nucleotide sequencecoding for a highly conserved region which may be identified by aligningthe different HPPD proteins.

“Stringent hybridization conditions” as used herein means thathybridization will generally occur if there is at least 95% andpreferably at least 97% sequence identity between the probe and thetarget sequence. Examples of stringent hybridization conditions areovernight incubation in a solution comprising 5×SSC (150 mM NaCl, 15 mMtrisodium-citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrierDNA such as salmon sperm DNA, followed by washing the hybridizationsupport in 0.1×SSC at approximately 65° C., preferably twice for about10 minutes. Other hybridization and wash conditions are well known andare exemplified in Sambrook et al, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularlychapter 11.

Such variant sequences may also be obtained by DNA amplification usingoligonucleotides specific for HPPD genes encoding enzymes as primers,such as but not limited to oligonucleotides comprising about 20 to about50 consecutive nucleotides selected from the nucleotide sequences of SEQID Nos 1, 2, 3 or their complement.

The invention also encompasses variant HPPD enzymes which are amino acidsequences similar to the HPPD amino acid sequence of SEQ ID No. 4wherein one or more amino acids have been inserted, deleted orsubstituted. In the present context, variants of an amino acid sequencerefer to those polypeptides, enzymes or proteins which have a similarcatalytic activity as the amino acid sequences described herein,notwithstanding any amino acid substitutions, additions or deletionsthereto. Preferably the variant amino acid sequence has a sequenceidentity of at least about 80%, or 85 or 90% or 95% with the amino acidsequence of SEQ ID No. 4. Also preferably, a polypeptide comprising thevariant amino acid sequence has HPPD enzymatic activity. Methods todetermine HPPD enzymatic activity are well known in the art and includeassays as extensively described in WO 2009/144079 or in WO 2002/046387.

Substitutions encompass amino acid alterations in which an amino acid isreplaced with a different naturally-occurring or a non-conventionalamino acid residue. Such substitutions may be classified as“conservative”, in which an amino acid residue contained in an HPPDprotein of this invention is replaced with another naturally-occurringamino acid of similar character, for example Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln or Phe

Trp

Tyr. Substitutions encompassed by the present invention may also be“non-conservative”, in which an amino acid residue which is present inan HPPD protein of the invention is substituted with an amino acid withdifferent properties, such as a naturally-occurring amino acid from adifferent group (eg. substituting a charged or hydrophobic amino acidwith alanine. Amino acid substitutions are typically of single residues,but may be of multiple residues, either clustered or dispersed. Aminoacid deletions will usually be of the order of about 1-10 amino acidresidues, while insertions may be of any length. Deletions andinsertions may be made to the N-terminus, the C-terminus or be internaldeletions or insertions. Generally, insertions within the amino acidsequence will be smaller than amino- or carboxy-terminal fusions and ofthe order of 1 to 4 amino acid residues. “Similar amino acids”, as usedherein, refers to amino acids that have similar amino acid side chains,i.e. amino acids that have polar, non-polar or practically neutral sidechains. “Non-similar amino acids”, as used herein, refers to amino acidsthat have different amino acid side chains, for example an amino acidwith a polar side chain is non-similar to an amino acid with a non-polarside chain. Polar side chains usually tend to be present on the surfaceof a protein where they can interact with the aqueous environment foundin cells (“hydrophilic” amino acids). On the other hand, “non-polar”amino acids tend to reside within the center of the protein where theycan interact with similar non-polar neighbours (“hydrophobic” aminoacids”). Examples of amino acids that have polar side chains arearginine, asparagine, aspartate, cysteine, glutamine, glutamate,histidine, lysine, serine, and threonine (all hydrophilic, except forcysteine which is hydrophobic). Examples of amino acids that havenon-polar side chains are alanine, glycine, isoleucine, leucine,methionine, phenylalanine, proline, and tryptophan (all hydrophobic,except for glycine which is neutral).

Also encompassed by the present invention are antibodies whichspecifically recognize a HPPD enzyme according to the invention.

The invention also relates to the use, in a method for transformingplants, of a nucleic acid which encodes an HPPD according to theinvention as a marker gene or as a coding sequence which makes itpossible to confer to the plant tolerance to herbicides which are HPPDinhibitors, and the use of HPPD inhibitors on plants comprising anucleic acid sequence encoding an HPPD according to the invention. In anembodiment of this invention, in such use the HPPD inhibitors aretriketones or pyrazolinates, preferably tembotrione, mesotrione orsulcotrione, bicyclopyrone, and tefuryltrione. It is, of course,understood that this sequence can also be used in combination with (an)other gene marker(s) and/or sequence(s) which encode(s) one or moreprotein with useful agricultural properties.

In the commercial production of crops, it is desirable to eliminateunder reliable pesticidial management unwanted plants (i.e., “weeds”)from a field of crop plants. An ideal treatment would be one which couldbe applied to an entire field but which would eliminate only theunwanted plants while leaving the crop plants unaffected. One suchtreatment system would involve the use of crop plants which are tolerantto an herbicide so that when the herbicide is sprayed on a field ofherbicide-tolerant crop plants, the crop plants would continue to thrivewhile non-herbicide-tolerant weeds are killed or severely damaged.Ideally, such treatment systems would take advantage of varyingherbicide properties so that weed control could provide the bestpossible combination of flexibility and economy. For example, individualherbicides have different longevities in the field, and some herbicidespersist and are effective for a relatively long time after they areapplied to a field while other herbicides are quickly broken down intoother and/or non-active compounds. An ideal treatment system would allowthe use of different herbicides so that growers could tailor the choiceof herbicides for a particular situation.

While a number of herbicide-tolerant crop plants are presentlycommercially available, one issue that has arisen for many commercialherbicides and herbicide/crop combinations is that individual herbicidestypically have incomplete spectrum of activity against common weedspecies. For most individual herbicides which have been in use for sometime, populations of herbicide resistant weed species and biotypes havebecome more prevalent (see, e.g., Tranel and Wright (2002) Weed Science50: 700-712; Owen and Zelaya (2005) Pest Manag. Sci. 61: 301-311).Transgenic plants which are resistant to more than one herbicide havebeen described (see, e.g., WO2005/012515). However, improvements inevery aspect of crop production, weed control options, extension ofresidual weed control, and improvement in crop yield are continuously indemand.

The HPPD protein or gene of the invention is advantageously combined inplants with other genes which encode proteins or RNAs that confer usefulagronomic properties to such plants. Among the genes which encodeproteins or RNAs that confer useful agronomic properties on thetransformed plants, mention can be made of the DNA sequences encodingproteins which confer tolerance to one or more herbicides that,according to their chemical structure, differ from HPPD inhibitorherbicides, and others which confer tolerance to certain insects, thosewhich confer tolerance to certain diseases, DNAs that encodes RNAs thatprovide nematode or insect control, etc. . . . .

Such genes are in particular described in published PCT PatentApplications WO 91/02071 and WO095/06128.

Among the DNA sequences encoding proteins which confer tolerance tocertain herbicides on the transformed plant cells and plants, mentioncan be made of a bar or PAT gene or the Streptomyces coelicolor genedescribed in WO2009/152359 which confers tolerance to glufosinateherbicides, a gene encoding a suitable EPSPS which confers tolerance toherbicides having EPSPS as a target, such as glyphosate and its salts(U.S. Pat. No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat. No.5,094,945, U.S. Pat. No. 4,940,835, U.S. Pat. No. 5,188,642, U.S. Pat.No. 4,971,908, U.S. Pat. No. 5,145,783, U.S. Pat. No. 5,310,667, U.S.Pat. No. 5,312,910, U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435),or a gene encoding glyphosate oxydoreductase (U.S. Pat. No. 5,463,175).

Among the DNA sequences encoding a suitable EPSPS which confer toleranceto the herbicides which have EPSPS as a target, mention will moreparticularly be made of the gene which encodes a plant EPSPS, inparticular maize EPSPS, particularly a maize EPSPS which comprises twomutations, particularly a mutation at amino acid position 102 and amutation at amino acid position 106 (WO 2004/074443), and which isdescribed in U.S. Pat. No. 6,566,587, hereinafter named double mutantmaize EPSPS or 2mEPSPS, or the gene which encodes an EPSPS isolated fromAgrobacterium and which is described by SEQ ID No. 2 and SEQ ID No. 3 ofU.S. Pat. No. 5,633,435, also named CP4.

Among the DNA sequences encoding a suitable EPSPS which confer toleranceto the herbicides which have EPSPS as a target, mention will moreparticularly be made of the gene which encodes an EPSPS GRG23 fromArthrobacter globiformis, but also the mutants GRG23 ACE1, GRG23 ACE2,or GRG23 ACE3, particularly the mutants or variants of GRG23 asdescribed in WO2008/100353, such as GRG23(ace3)R173K of SEQ ID No. 29 inWO2008/100353.

In the case of the DNA sequences encoding EPSPS, and more particularlyencoding the above genes, the sequence encoding these enzymes isadvantageously preceded by a sequence encoding a transit peptide, inparticular the “optimized transit peptide” described in U.S. Pat. Nos.5,510,471 or 5,633,448.

In WO 2007/024782, plants being tolerant to glyphosate and at least oneALS (acetolactate synthase) inhibitor are disclosed. More specificallyplants containing genes encoding a GAT (Glyphosate-N-Acetyltransferase)polypeptide and a polypeptide conferring resistance to ALS inhibitorsare disclosed. In U.S. Pat. No. 6,855,533, transgenic tobacco plantscontaining mutated Arabidopsis ALS/AHAS genes were disclosed.

In U.S. Pat. No. 6,153,401, plants containing genes encoding2,4-D-monooxygenases conferring tolerance to 2,4-D(2,4-dichlorophenoxyacetic acid) by metabolisation are disclosed.

In US 2008/0119361 and US 2008/0120739, plants containing genes encodingDicamba monooxygenases conferring tolerance to dicamba(3,6-dichloro-2-methoxybenzoic acid) by metabolisation are disclosed.

All the above mentioned herbicide tolerance traits can be combined withthose performing HPPD tolerance which are subject matter of thisinvention.

Among the DNA sequences encoding proteins concerning properties oftolerance to insects, mention will more particularly be made of the Btproteins widely described in the literature and well known to thoseskilled in the art. Mention will also be made of proteins extracted frombacteria such as Photorhabdus (WO 97/17432 & WO 98/08932).

Among such DNA sequences encoding proteins of interest which confernovel properties of tolerance to insects, mention will more particularlybe made of the Bt Cry or VIP proteins widely described in the literatureand well known to those skilled in the art. These include the Cry1Fprotein or hybrids derived from a Cry1F protein (e.g., the hybridCry1A-Cry1F proteins described in U.S. Pat. No. 6,326,169; U.S. Pat. No.6,281,016; U.S. Pat. No. 6,218,188, or toxic fragments thereof), theCry1A-type proteins or toxic fragments thereof, preferably the Cry1Acprotein or hybrids derived from the Cry1Ac protein (e.g., the hybridCry1Ab-Cry1Ac protein described in U.S. Pat. No. 5,880,275) or theCry1Ab or Bt2 protein or insecticidal fragments thereof as described inEP451878, the Cry2Ae, Cry2Af or Cry2Ag proteins as described inWO02/057664 or toxic fragments thereof, the Cry1A.105 protein describedin WO 2007/140256 (SEQ ID No. 7) or a toxic fragment thereof, theVIP3Aa19 protein of NCBI accession ABG20428, the VIP3Aa20 protein ofNCBI accession ABG20429 (SEQ ID No. 2 in WO 2007/142840), the VIP3Aproteins produced in the COT202 or COT203 cotton events (WO 2005/054479and WO 2005/054480, respectively), the Cry proteins as described inWO01/47952, the VIP3Aa protein or a toxic fragment thereof as describedin Estruch et al. (1996), Proc Natl Acad Sci USA. 28; 93 (11):5389-94and U.S. Pat. No. 6,291,156, the insecticidal proteins from Xenorhabdus(as described in WO98/50427), Serratia (particularly from S.entomophila) or Photorhabdus species strains, such as Tc-proteins fromPhotorhabdus as described in WO98/08932 (e.g., Waterfield et al., 2001,Appl Environ Microbiol. 67 (11):5017-24; Ffrench-Constant and Bowen,2000, Cell Mol Life Sci.; 57 (5):828-33). Also any variants or mutantsof any one of these proteins differing in some (1-10, preferably 1-5)amino acids from any of the above sequences, particularly the sequenceof their toxic fragment, or which are fused to a transit peptide, suchas a plastid transit peptide, or another protein or peptide, is includedherein.

The present invention also relates to a chimeric gene (or expressioncassette) which comprises a coding sequence as well as heterologousregulatory elements, at the 5′ and/or 3′ position, at least at the 5′position, which are able to function in a host organism, in particularplant cells or plants, with the coding sequence containing at least onenucleic acid sequence which encodes an HPPD as previously defined.

In a particular embodiment, the present invention relates to a chimericgene as previously described, wherein the host organism is selected frombacteria, yeast, Pichia, fungi, baculovirus, in vitro cells,protoplasts, plant cells, plants, plant parts, and plant seeds thereof.

In another particular embodiment, the present invention relates to achimeric gene as previously described, wherein the chimeric genecontains in the 5′ position of the nucleic acid sequence which encodes aHPPD according to the invention, a nucleic acid sequence which encodes aplant transit peptide, with this sequence being arranged between thepromoter region and the sequence encoding the HPPD according to theinvention so as to permit expression of a transit peptide/HPPD fusionprotein.

In a further particular embodiment, the present invention relates to theuse of HPPD inhibitor herbicides on plants, plant parts, or plant seedscomprising HPPD tolerant gene according to the invention, or to the useof HPPD inhibitor herbicides on soil where such plants, plant parts orseeds are to be grown or sown, either alone or in combination with oneor more other known herbicides acting in a different matter to HPPDinhibitors. In a more particular embodiment, the employed HPPD inhibitorherbicide is selected from the group consisting of triketones (namedtriketone HPPD inhibitor), such as tembotrione, sulcotrione mesotrione,bicyclopyrone, tefuryltrione, particularly tembotrione, of the classdiketone such as diketonitrile of the class of isoxazoles such asisoxaflutole or of the class of pyrazolinates (named pyrazolinate HPPDinhibitor), such as pyrasulfotole, pyrazolate, topramezone, benzofenap,even more specifically present invention relates to the application oftembotrione, mesotrione, diketonitrile, bicyclopyrone, tefuryltrione,benzofenap, pyrasulfotole, pyrazolate and sulcotrione to such HPPDinhibitor tolerant plants, plant parts or plant seeds.

As a regulatory sequence which functions as a promoter in plant cellsand plants, use may be made of any promoter sequence of a gene which isnaturally expressed in plants, in particular a promoter which isexpressed especially in the leaves of plants, such as for example“constitutive” promoters of bacterial, viral or plant origin, or“light-dependent” promoters, such as that of a plantribulose-biscarboxylase/oxygenase (RuBisCO) small subunit gene, or anysuitable known promoter-expressible which may be used. Among thepromoters of plant origin, mention will be made of the histone promotersas described in EP 0 507 698 A1, the rice actin promoter (U.S. Pat. No.5,641,876), or a plant ubiquitin promoter (U.S. Pat. No. 5,510,474).Among the promoters of a plant virus gene, mention will be made of thatof the cauliflower mosaic virus (CaMV 19S or 35S, Sanders et al. (1987),Nucleic Acids Res. 15 (4):1543-58), the circovirus (AU 689 311) or theCassava vein mosaic virus (CsVMV, U.S. Pat. No. 7,053,205).

In one embodiment of this invention, a promoter sequence specific forparticular regions or tissues of plants can be used to express the HPPDproteins of the invention, such as promoters specific for seeds (Datla,R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially thenapin promoter (EP 255 378 A1), the phaseolin promoter, the gluteninpromoter, the helianthinin promoter (WO 92/17580), the albumin promoter(WO 98/45460), the oleosin promoter (WO 98/45461), the SAT1 promoter orthe SAT3 promoter (PCT/US98/06978).

Use may also be made of an inducible promoter advantageously chosen fromthe phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG),chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene,nopaline synthase (nos) and vspB promoters (U.S. Pat. No. 5,670,349,Table 3), the HMG2 promoter (U.S. Pat. No. 5,670,349), the applebeta-galactosidase (ABG1) promoter and the apple aminocyclopropanecarboxylate synthase (ACC synthase) promoter (WO 98/45445).

According to the invention, use may also be made, in combination withthe promoter, of other regulatory sequences, which are located betweenthe promoter and the coding sequence, such as transcription activators(“enhancers”), for instance the translation activator of the tobaccomosaic virus (TMV) described in Application WO 87/07644, or of thetobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol.64: 1590-1597, for example, or introns such as the adh1 intron of maizeor intron 1 of rice actin.

In a further particular embodiment, the gene of the invention is presentin plants in multiple, preferably two copies, each of these controlledby a different plant expressible promoter.

In a further particular embodiment, the chimeric gene of the inventioncan be combined with any further chimeric gene coding for an HPPDprotein, preferably these different genes are controlled by differentregulatory elements being active in plants.

In a further particular embodiment, the chimeric gene of the inventioncan be combined with a CYP450 Maize monooxygenase (nsf1 gene) gene beingunder the control of an identical or different plant expressiblepromoter.

As a regulatory terminator or polyadenylation sequence, use may be madeof any corresponding sequence of bacterial origin, such as for examplethe nos terminator of Agrobacterium tumefaciens, of viral origin, suchas for example the CaMV 35S terminator, or of plant origin, such as forexample a histone terminator as described in published PatentApplication EP 0 633 317 A1.

The term “gene”, as used herein refers to a DNA coding region flanked by5′ and/or 3′ regulatory sequences allowing a RNA to be transcribed whichcan be translated to a protein, typically comprising at least a promoterregion. A “chimeric gene”, when referring to an HPPD encoding DNA ofthis invention, refers to an HPPD encoding DNA sequence having 5′ and/or3′ regulatory sequences different from the naturally occurringEuryarchaeota 5′ and/or 3′ regulatory sequences which drive theexpression of the HPPD protein in its native host cell (also referred toas “heterologous promoter” or “heterologous regulatory sequences”). Theterms “DNA/protein comprising the sequence X” and “DNA/protein with thesequence comprising sequence X”, as used herein, refer to a DNA orprotein including or containing at least the sequence X in theirnucleotide or amino acid sequence, so that other nucleotide or aminoacid sequences can be included at the 5′ (or N-terminal) and/or 3′ (orC-terminal) end, e.g., a N-terminal transit or signal peptide. The term“comprising”, as used herein, is open-ended language in the meaning of“including”, meaning that other elements then those specifically recitedcan also be present. The term “consisting of”, as used herein, isclosed-ended language, i.e., only those elements specifically recitedare present. The term “DNA encoding a protein comprising sequence X”, asused herein, refers to a DNA comprising a coding sequence which aftertranscription and translation results in a protein containing at leastamino acid sequence X. A DNA encoding a protein need not be a naturallyoccurring DNA, and can be a semi-synthetic, fully synthetic orartificial DNA and can include introns and 5′ and/or 3′ flankingregions. The term “nucleotide sequence”, as used herein, refers to thesequence of a DNA or RNA molecule, which can be in single- ordouble-stranded form.

HPPD proteins according to the invention may be equipped with a signalpeptide according to procedures known in the art, see, e.g., publishedPCT patent application WO 96/10083, or they can be replaced by anotherpeptide such as a chloroplast transit peptide (e.g., Van Den Broeck etal., 1985, Nature 313, 358, or a modified chloroplast transit peptide ofU.S. Pat. No. 5,510,471) causing transport of the protein to thechloroplasts, by a secretory signal peptide or a peptide targeting theprotein to other plastids, mitochondria, the ER, or another organelle,or it can be replaced by a methionine amino acid or by amethionine-alanine dipeptide. Signal sequences for targeting tointracellular organelles or for secretion outside the plant cell or tothe cell wall are found in naturally targeted or secreted proteins,preferably those described by Klosgen et al. (1989, Mol. Gen. Genet.217, 155-161), Klosgen and Weil (1991, Mol. Gen. Genet. 225, 297-304),Neuhaus & Rogers (1998, Plant Mol. Biol. 38, 127-144), Bih et al. (1999,J. Biol. Chem. 274, 22884-22894), Morris et al. (1999, Biochem. Biophys.Res. Commun. 255, 328-333), Hesse et al. (1989, EMBO J. 8 2453-2461),Tavladoraki et al. (1998, FEBS Lett. 426, 62-66), Terashima et al.(1999, Appl. Microbiol. Biotechnol. 52, 516-523), Park et al. (1997, J.Biol. Chem. 272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad.Sci USA 92, 9245-9249), all of which are incorporated herein byreference, particularly the signal peptide sequences from targeted orsecreted proteins of corn, cotton, soybean, or rice. A DNA sequenceencoding such a plant signal peptide can be inserted in the chimericgene encoding the HPPD protein for expression in plants

Unless otherwise stated in the examples, all procedures for making andmanipulating recombinant DNA are carried out by the standard proceduresdescribed in Sambrook et al., Molecular Cloning—A Laboratory Manual,Second Ed., Cold Spring Harbor Laboratory Press, NY (1989), and inVolumes 1 and 2 of Ausubel et al. (1994) Current Protocols in MolecularBiology, Current Protocols, USA. Standard materials and methods forplant molecular biology work are described in Plant Molecular BiologyLabfax (1993) by R. R. D. Croy, jointly published by BIOS ScientificPublications Ltd (UK) and Blackwell Scientific Publications (UK).Procedures for PCR technology can be found in “PCR protocols: a guide tomethods and applications”, Edited by M. A. Innis, D. H. Gelfand, J. J.Sninsky and T. J. White (Academic Press, Inc., 1990).

The terms “tolerance”, “tolerant” or “less sensitive” areinterchangeable used and mean the relative levels of inherent toleranceof the HPPD screened according to a visible indicator phenotype of thestrain or plant transformed with a nucleic acid comprising the genecoding for the respective HPPD protein in the presence of differentconcentrations of the various HPPD inhibitors. Dose responses andrelative shifts in dose responses associated with these indicatorphenotypes (formation of brown colour, growth inhibition, bleaching,herbicidal effect etc) are conveniently expressed in terms, for example,of GR50 (concentration for 50% reduction of growth) or MIC (minimuminhibitory concentration) values where increases in values correspond toincreases in inherent tolerance of the expressed HPPD, in the normalmanner based upon plant damage, meristematic bleaching symptoms etc. ata range of different concentrations of herbicides. These data can beexpressed in terms of, for example, GR50 values derived fromdose/response curves having “dose” plotted on the x-axis and “percentagekill”, “herbicidal effect”, “numbers of emerging green plants” etc.plotted on the y-axis where increased GR50 values correspond toincreased levels of inherent tolerance of the expressed HPPD. Herbicidescan suitably be applied pre-emergence or post emergence.

Likewise, tolerance level of the nucleic acid or gene encoding an HPPDprotein according to the invention, or the HPPD protein of the inventionis screened via transgenesis, regeneration, breeding and spray testingof a test plant such as tobacco, or a crop plant such as soybean orcotton and according to these results, such plants are at least 2-4×more tolerant to HPPD inhibitors like tembotrione, mesotrione,diketonitrile and/or bicyclopyrone, than plants that do not contain anyexogenous gene encoding an HPPD protein, or than plants that contain agene comprising an Arabidopsis thaliana HPPD-encoding DNA, under controlof the same promoter as the HPPD DNA of the invention.

“Host organism” or “host” is understood as being any unicellular ormulticellular heterologous organism into which the nucleic acid orchimeric gene according to the invention can be introduced for thepurpose of producing HPPD according to the invention. These organismsare, in particular, bacteria, for example E. coli, yeasts, in particularof the genera Saccharomyces or Kluyveromyces, Pichia, fungi, inparticular Aspergillus, a baculovirus or, preferably, plant cells andplants.

“Plant cell” is understood, according to the invention, as being anycell which is derived from or found in a plant and which is able to formor is part of undifferentiated tissues, such as calli, differentiatedtissues such as embryos, parts of plants, plants or seeds. This includesprotoplasts and pollen, cultivated plants cells or protoplasts grown invitro, and plant cells that can regenerate into a complete plant.

“Plant” is understood, according to the invention, as being anydifferentiated multicellular organism which is capable ofphotosynthesis, in particular a monocotyledonous or dicotyledonousorganism, more especially cultivated plants which are or are notintended for animal or human nutrition, such as maize or corn, wheat,Brassica spp. plants such as Brassica napus or Brassica juncea, soyaspp, rice, sugarcane, beetroot, tobacco, cotton, vegetable plants suchas cucumber, leek, carrot, tomato, lettuce, peppers, melon, watermelon,etc. Transgenic plants, as used herein, refer to plants comprising aforeign or heterologous gene stably inserted in their genome.

In one embodiment the invention relates to the transformation of plants.Any promoter sequence of a gene which is expressed naturally in plants,or any hybrid or combination of promoter elements of genes expressednaturally in plants, including Agrobacterium or plant virus promoters,or any promoter which is suitable for controlling the transcription of aherbicide tolerance gene in plants, can be used as the promoter sequencein the plants of the invention (named “plant-expressible promoter”herein). Examples of such suitable plant-expressible promoters aredescribed above. In one embodiment of this invention, suchplant-expressible promoters are operably-linked to a coding sequenceencoding an HPPD protein of the invention to form a chimeric HPPD geneof this invention.

According to the invention, it is also possible to use, in combinationwith the promoter regulatory sequence, other regulatory sequences whichare located between the promoter and the coding sequence, such as intronsequences, or transcription activators (enhancers). Examples of suchsuitable regulatory sequences are described above.

Any corresponding sequence of bacterial or viral origin, such as the nosterminator from Agrobacterium tumefaciens, or of plant origin, such as ahistone terminator as described in application EP 0 633 317 A1, may beused as transcription termination (and polyadenylation) regulatorysequence.

In one particular embodiment of the invention, a nucleic acid sequencewhich encodes a transit peptide is employed 5′ (upstream) of the nucleicacid sequence encoding the exogenous HPPD according to the invention,with this transit peptide sequence being arranged between the promoterregion and the sequence encoding the exogenous HPPD so as to permitexpression of a transit peptide-HPPD fusion protein, such as the proteinof SEQ ID No. 6 or SEQ ID No. 7. The transit peptide makes it possibleto direct the HPPD into the plastids, more especially the chloroplasts,with the fusion protein being cleaved between the transit peptide andthe HPPD protein of the invention when the latter enters the plastid.The transit peptide may be a single peptide, such as an EPSPS transitpeptide (described in U.S. Pat. No. 5,188,642) or a transit peptide ofthe plant ribulose bisphosphate carboxylase/oxygenase small subunit(RuBisCO ssu), where appropriate, including a few amino acids of theN-terminal part of the mature RuBisCO ssu (EP 189 707 A1), or else maybe a fusion of several transit peptides such as a transit peptide whichcomprises a first plant transit peptide which is fused to a part of theN-terminal sequence of a mature protein having a plastid location, withthis part in turn being fused to a second plant transit peptide asdescribed in patent EP 508 909 A1, and, more especially, the optimizedtransit peptide which comprises a transit peptide of the sunflowerRuBisCO ssu fused to 22 amino acids of the N-terminal end of the maizeRuBisCO ssu, in turn fused to the transit peptide of the maize RuBisCOssu, as described, with its coding sequence, in patent EP 508 909 A1.The present invention also relates to the transit peptide-HPPD fusionprotein and a nucleic acid or plant-expressible chimeric gene encodingsuch fusion protein, wherein the two elements of this fusion protein areas defined above.

The present invention also relates to a cloning, transformation and/orexpression vector, which vector contains at least one chimeric gene asdefined above. In addition to the above chimeric gene, this vector cancontain an origin of replication. This vector can be a plasmid orplasmid portion, a cosmid, or a bacteriophage or a virus which has beentransformed by introducing the chimeric gene according to the invention.Transformation vectors are well known to the skilled person and widelydescribed in the literature. The transformation vector which can beused, in particular, for transforming plant cells or plants may be avirus, which can be employed for transforming plant cells or plants andwhich additionally contains its own replication and expression elements.According to the invention, the vector for transforming plant cells orplants is preferably a plasmid, such as a disarmed Agrobacterium Tiplasmid.

The present invention also relates to the host organisms, in particularplant cells, seeds or plants, which comprise a chimeric gene whichcomprises a sequence encoding an HPPD protein of the invention, such asa protein comprising the amino acid sequence of SEQ ID Nos 4, 5, 6, or 7as defined above, and the use of the plants or seeds of the invention ina field to grow a crop and harvest a plant product, e.g., soya spp,rice, wheat, barley or corn grains or cotton bolls, where in oneembodiment said use involves the application of an HPPD inhibitorherbicide to such plants to control weeds. In one embodiment of thisinvention, in such use the HPPD inhibitors are triketones orpyrazolinates, preferably tembotrione, mesotrione, topramezone orsulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap andtefuryltrione, particularly tembotrione.

Therefore, the present invention relates to a host organism, inparticular a plant cell, seed, or plant, characterized in that itcontains at least one HPPD chimeric gene as described above, or at leastan HPPD nucleic acid sequence as previously described.

In a particular embodiment, the present invention relates to a plantcell or plant characterized in that it contains at least a nucleic acidsequence which encodes an HPPD protein of this invention which retainits properties of catalysing the conversion ofpara-hydroxyphenylpyruvate to homogentisate and which makes this plantmore tolerant than plants of the same species not comprising such HPPDprotein of the present invention, particularly to triketones, orpyrazolinates, preferably tembotrione, mesotrione, topramezone orsulcotrione, bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap andtefuryltrione, particularly tembotrione and such plants containing theHPPD of the invention have an agronomically acceptable tolerance to anHPPD inhibitor herbicide particularly to triketones, or pyrazolinates,preferably tembotrione, mesotrione, topramezone or sulcotrione,bicyclopyrone, pyrasulfotole, pyrazolate, benzofenap and tefuryltrione,particularly tembotrione.

In another particular embodiment, the present invention relates to aplant cell or plant characterized in that it contains at least a nucleicacid sequence which encodes an HPPD of this invention which retain itsproperties of catalysing the conversion of para-hydroxyphenylpyruvate tohomogentisate and which is less sensitive to an HPPD inhibitor than thehost plant endogenous HPPD, such as the HPPD from Arabidopsis thaliana,particularly the HPPD comprising the amino acid sequence of SEQ ID No.11 (from the amino acid position 126 to the amino acid position 568), orcomprising the amino acid sequence of SEQ ID No. 11 or SEQ ID No. 12(from the amino acid position 134 to the amino acid position 575).

In a particular embodiment, the present invention relates to a hostplant cell, seed or host plant characterized in that it contains atleast a nucleic acid sequence which encodes an HPPD of the invention asdefined herein, wherein the HPPD of the invention is less sensitive thanthe host plant endogenous HPPD to an HPPD inhibitor herbicide of theclass of isoxazoles, diketonitriles, triketones or pyrazolinates moreespecially from isoxaflutole, tembotrione, mesotrione, sulcotrione,pyrasulfotole, bicyclopyrone, tefuryltrione, topramezone,2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3 Cl₂ phenyl)propane-1,3-dione,even more particularly tembotrione, mesotrione, diketonitrile,bicyclopyrone, topramezone, pyrazolate, benzofenap, sulcotrione,tefuryltrione, and pyrasulfotole, most particularly tembotrione,mesotrione and bicyclopyrone.

In another particular embodiment, the present invention relates to aplant cell or plant characterized in that it contains at least a nucleicacid sequence encoding an HPPD of the invention as previously described,and in addition a chimeric gene comprising a plant-expressible promoteras described above, operably-linked to a nucleic acid sequence encodinga PDH (prephenate dehydrogenase) enzyme (US 2005/0257283).

The present invention also relates to the plants which containtransformed cells, in particular the plants which are regenerated fromthe transformed cells, and progeny plants or seeds thereof, comprisingthe chimeric HPPD gene of the invention. The regeneration can beobtained by any appropriate method, with the method depending on thenature of the species, as described, for example, in the abovereferences. The following patents and patent applications may be cited,in particular, with regard to the methods for transforming plant cellsand regenerating plants: U.S. Pat. No. 4,459,355, U.S. Pat. No.4,536,475, U.S. Pat. No. 5,464,763, U.S. Pat. No. 5,177,010, U.S. Pat.No. 5,187,073, EP 267,159 A1, EP 604 662 A1, EP 672 752 A1, U.S. Pat.No. 4,945,050, U.S. Pat. No. 5,036,006, U.S. Pat. No. 5,100,792, U.S.Pat. No. 5,371,014, U.S. Pat. No. 5,478,744, U.S. Pat. No. 5,179,022,U.S. Pat. No. 5,565,346, U.S. Pat. No. 5,484,956, U.S. Pat. No.5,508,468, U.S. Pat. No. 5,538,877, U.S. Pat. No. 5,554,798, U.S. Pat.No. 5,489,520, U.S. Pat. No. 5,510,318, U.S. Pat. No. 5,204,253, U.S.Pat. No. 5,405,765, EP 442 174 A1, EP 486 233 A1, EP 486 234 A1, EP 539563 A1, EP 674 725 A1, WO 91/02071 and WO 95/06128.

The present invention also relates to the transgenic plants or partthereof, which are derived by cultivating and/or crossing the abovetransgenic plants, and to the seeds of the transgenic plants, comprisingthe HPPD chimeric gene of the invention.

The present invention also relates to the end products such as the mealor oil which are obtained from the plants, part thereof, or seeds of theinvention.

The transformed plants which can be obtained in accordance with theinvention can be of the monocotyledonous type, such as wheat, barley,sugarcane, rice, onion, and corn or maize, or of the dicotyledonoustype, such as tobacco, soya spp, alfalfa Brassica spp. plants such asoilseed rape, cotton, sugarbeet clover, vegetables, etc.

The invention relates to a method for transforming host organisms, inparticular plant cells or plants, by integrating in such organisms atleast one nucleic acid sequence or one chimeric gene as previouslydefined, wherein it is possible to obtain the transformation by anyappropriate known means, which means are amply described in thespecialist literature and, in particular, the references cited in thepresent application, e.g., by using the vector according to theinvention.

One transformation method in accordance with this invention comprisesbombarding cells, protoplasts or tissues with solid or liquid particlesto which DNA is attached, or containing DNA. Another transformationmethod comprises using, as mean for transfer into the plant, a chimericgene which is inserted into an Agrobacterium tumefaciens Ti plasmid oran Agrobacterium rhizogenes Ri plasmid. Other methods may be used, suchas microinjection or electroporation or otherwise direct gene transferusing PEG. The skilled person can select any appropriate method fortransforming the host organism of choice, in particular the plant cellor the plant. As examples, the technology for soybean transformation hasbeen extensively described in the examples 1 to 3 disclosed in EP1186666 A1, incorporated herein by reference. For rice,Agrobacterium-mediated transformation (Hiei et al., 1994 Plant J6:271-282, and Hiei et al., 1997 Plant Mol Biol. 35:205-21, incorporatedherein by reference), electroporation (U.S. Pat. No. 5,641,664 and U.S.Pat. No. 5,679,558, incorporated herein by reference), or bombardment(Christou et al., 1991, Biotechnology 9:957 incorporated herein byreference) could be performed. A suitable technology for transformationof monocotyledonous plants, and particularly rice, is described in WO92/09696, incorporated herein by reference. For cotton,Agrobacterium-mediated transformation (Gould J. H. and Magallanes-CedenoM., 1998 Plant Molecular Biology reporter, 16:1-10 and Zapata C., 1999,Theoretical Applied Genetics, 98(2):1432-2242 incorporated herein byreference), polybrene and/or treatment-mediated transformation (SawahelW. A., 2001,—Plant Molecular Biology reporter, 19:377a-377f,incorporated herein by reference) have been described.

In a particular embodiment of the invention, the HPPD of the inventionis targeted into the chloroplast. This may be done by fusing a nucleicacid sequence which encodes a transit peptide to the nucleic acidsequence encoding the HPPD protein of the invention to obtain a nucleicacid encoding a fusion protein as described above. Alternatively, theHPPD of the invention may be expressed directly in the plastids, such asthe chloroplasts, using transformation of the plastid, such as thechloroplast genome. A suitable method comprises the bombardment of plantcells or tissue by solid particles coated with the DNA or liquidparticles comprising the DNA, and integration of the introduced geneencoding the protein of the invention by homologous recombination.Suitable vectors and selection systems are known to the person skilledin the art. An example of means and methods which can be used for suchintegration into the chloroplast genome of tobacco plants is given in WO06/108830, the content of which is hereby incorporated by reference

The present invention also relates to a method for obtaining a plant toan HPPD inhibitor, characterized in that the plant is transformed with achimeric HPPD gene of the invention as previously described.

Therefore, the present invention also relates to a method for obtaininga plant tolerant to an HPPD inhibitor, characterized in that the plantcontains a chimeric HPPD gene of the invention which comprises a codingsequence as well as a heterologous regulatory element in the 5′ andoptionally in the 3′ positions, which are able to function in a hostorganism, characterized in that the coding sequence comprises at least anucleic acid sequence defining a gene encoding an HPPD of the inventionas previously described.

In one embodiment of this invention, the HPPD inhibitor in the abovemethod is a triketone or pyrazolinate herbicide, preferably tembotrione,mesotrione, bicyclopyrone, tefuryltrione pyrasulfotole, pyrazolate,diketonitrile, benzofenap, or sulcotrione, particularly tembotrione.

According to this invention, a method for obtaining a plant tolerant toan HPPD inhibitor as described above is also provided, characterized inthat a plant is obtained comprising a first transgene which is achimeric HPPD gene of the invention, and a second transgene, which is achimeric gene comprising a plant-expressible promoter operably-linked toa nucleic acid encoding a PDH (prephenate dehydrogenase) enzyme. A plantcomprising such two transgenes can be obtained by transforming a plantwith one transgene, and then re-transforming this transgenic plant withthe second transgene, or by transforming a plant with the two transgenessimultaneously (in the same or in 2 different transforming DNAs orvectors), or by crossing a plant comprising the first transgene with aplant comprising the second transgene, as is well known in the art.

The invention also relates to a method for selectively removing weeds orpreventing the germination of weeds in a field to be planted with plantsor to be sown with seeds, or in a plant crop, by application of an HPPDinhibitor to such field or plant crop, in particular an HPPD inhibitorheribicide as previously defined, which method is characterized in thatthis HPPD inhibitor herbicide is applied to plants which have beentransformed in accordance with the invention, either before sowing thecrop (hereinafter named pre-planting application), before emergence ofthe crop (hereinafter named pre-emergence application), or afteremergence of the crop (hereinafter named post-emergence application).

The invention also relates to a method for controlling in an area or afield which contains transformed seeds as previously described in thepresent invention, which method comprises applying, to the said area ofthe field, a dose of an HPPD inhibitor herbicide which is toxic for thesaid weeds, without significantly affecting the seeds or plants whichcontain the HPPD nucleic acid or the chimeric HPPD gene of the inventionas previously described in the present invention.

The present invention also relates to a method for cultivating theplants which have been transformed with a chimeric gene according to theinvention, which method comprises planting seeds comprising a chimericgene of the invention, in an area of a field which is appropriate forcultivating the said plants, and in applying, if weeds are present, adose, which is toxic for the weeds, of a herbicide whose target is theabove-defined HPPD to the said area of the said field, withoutsignificantly affecting the said transformed seeds or the saidtransformed plants, and in then harvesting the cultivated plants orplant parts when they reach the desired stage of maturity and, whereappropriate, in separating the seeds from the harvested plants.

In the above methods, the herbicide whose target is the HPPD enzyme canbe applied in accordance with the invention, either before sowing thecrop, before the crop emerges or after the crop emerges.

The present invention also relates to a process for obtaining oil,particularly soya spp, corn, or cotton oil, or meal, comprising growinga crop, particularly a soya spp crop, expressing an HPPD protein of theinvention optionally treating such crop with an HPPD inhibitorherbicide, harvesting the grains and milling the grains to make meal andextract the oil. Also the seeds or grains, either whole, broken orcrushed, comprising the chimeric gene of the invention are part of thisinvention.

Therefore, the present invention relates to a method for obtaining oilor meal comprising growing a transformed plant as described above,optionally treating such plant with an HPPD inhibitor herbicide,harvesting the grains and milling the grains to make meal and extractthe oil.

Further provided in this invention, are the above methods involving anHPPD inhibitor herbicide selected from isoxaflutole, tembotrione,mesotrione, pyrasulfotole, sulcotrione, bicyclopyrone, tefuryltrione,topramezone,2-cyano-3-cyclopropyl-1-(2-methylsulphonyl-4-trifluoromethylphenyl)-propane-1,3-dioneand to2-cyano-1-[4-(methylsulphonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione.

Also provided herein are the above methods of the invention involving anHPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione.

Within the meaning of the present invention, “herbicide” is understoodas being a herbicidally active substance on its own or such a substancewhich is combined with an additive which alters its efficacy, such as,for example, an agent which increases its activity (a synergistic agent)or which limits its activity (a safener). It is of course to beunderstood that, for their application in practice, the above herbicidesare combined, in a manner which is known per se, with the formulationadjuvants which are customarily employed in agricultural chemistry.

HPPD inhibitor herbicides like those of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione, have anoutstanding herbicidal activity against a broad spectrum of economicallyimportant monocotyledonous and dicotyledonous annual harmful plants. Theactive substances also act efficiently on perennial harmful plants whichproduce shoots from rhizomes, wood stocks or other perennial organs andwhich are difficult to control.

The present invention therefore also relates to a method of controllingundesired plants or for regulating the growth of plants in crops ofplants comprising an HPPD according to the invention, where one or moreHPPD inhibitor herbicides of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione are appliedto the plants (for example harmful plants such as monocotyledonous ordicotyledonous weeds or undesired crop plants), to the seeds (forexample grains, seeds or vegetative propagules such as tubers or shootparts with buds) or to the area on which the plants grow (for examplethe area under cultivation). In this context, an HPPD inhibitorherbicide of the class of triketones, such as tembotrione, sulcotrioneand mesotrione, or of the class of pyrazolinates, such as pyrasulfotoleand topramezone, particularly selected from tembotrione, sulcotrione,topramezone, bicyclopyrone, tefuryltrione and mesotrione, moreparticularly tembotrione can be applied for example pre-planting (ifappropriate also by incorporation into the soil), pre-emergence orpost-emergence. Examples of individual representatives of themonocotyledonous and dicotyledonous weeds which can be controlled withan HPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione are herebymentioned, without this mentioning being intended as a limitation tocertain species only:

-   -   Monocotyledonous harmful plants of the genera: Aegilops,        Agropyron, Agrostis, Alopecurus, Apera, Avena, Brachiaria,        Bromus, Cenchrus, Commelina, Cynodon, Cyperus, Dactyloctenium,        Digitaria, Echinochloa, Eleocharis, Eleusine, Eragrostis,        Eriochloa, Festuca, Fimbristylis, Heteranthera, Imperata,        Ischaemum, Leptochloa, Lolium, Monochoria, Panicum, Paspalum,        Phalaris, Phleum, Poa, Rottboellia, Sagittaria, Scirpus,        Setaria, Sorghum.    -   Dicotyledonous weeds of the genera: Abutilon, Amaranthus,        Ambrosia, Anoda, Anthemis, Aphanes, Artemisia, Atriplex, Bellis,        Bidens, Capsella, Carduus, Cassia, Centaurea, Chenopodium,        Cirsium, Convolvulus, Datura, Desmodium, Emex, Erysimum,        Euphorbia, Galeopsis, Galinsoga, Galium, Hibiscus, Ipomoea,        Kochia, Lamium, Lepidium, Lindernia, Matricaria, Mentha,        Mercurialis, Mullugo, Myosotis, Papaver, Pharbitis, Plantago,        Polygonum, Portulaca, Ranunculus, Raphanus, Rorippa, Rotala,        Rumex, Salsola, Senecio, Sesbania, Sida, Sinapis, Solanum,        Sonchus, Sphenoclea, Stellaria, Taraxacum, Thlaspi, Trifolium,        Urtica, Veronica, Viola, Xanthium.

In transgenic crops according to the invention, comprising an HPPDprotein, DNA or chimeric gene according invention and which may alsoshow one more further herbicide resistances against herbicides thatdiffer from HPPD inhibitor herbicides, the use of HPPD inhibitorherbicide of the class of triketones, such as tembotrione, sulcotrioneand mesotrione, or of the class of pyrazolinates, such as pyrasulfotoleand topramezone, particularly selected from tembotrione, sulcotrione,topramezone, bicyclopyrone, tefuryltrione and mesotrione, moreparticularly tembotrione in economically important transgenic crops ofuseful plants and ornamentals, for example of cereals such as wheat,barley, rye, oats, sorghum and millet, rice and maize or else crops ofsugar beet, cotton, soya spp, oil seed rape, potato, tomato, peas andother vegetables is preferred.

As it relates to plant properties other than the tolerance to HPPDinhibitor herbicides as described in the present invention, conventionalways of generating novel plants which, in comparison with existingplants, have modified properties are, for example, traditional breedingmethods and the generation of mutants. Alternatively, novel plants withmodified properties can be generated with the aid of recombinant methods(see, for example, EP-A-0221044 A1, EP-A-0131624 A1). For example, thefollowing have been described in several cases:

-   -   recombinant modifications of crop plants for the purposes of        modifying the starch synthesized in the plants (for example WO        92/11376, WO 92/14827, WO 91/19806),    -   transgenic crop plants which are resistant to certain herbicides        of the glufosinate type (cf., for example, EP-A-0242236,        EP-A-242246) or of the glyphosate type (WO 92/00377) or of the        sulfonylurea type (EP-A-0257993, U.S. Pat. No. 5,013,659),    -   transgenic crop plants, for example corn, cotton or soya spp,        which are capable of producing Bacillus thuringiensis toxins (Bt        toxins), or hybrids or mutants thereof, which make the plants        resistant to certain pests (EP-A-0193259),    -   transgenic crop plants with a modified fatty acid composition        (WO 91/13972),    -   genetically modified crop plants with novel constituents or        secondary metabolites, for example novel phytoalexins, which        bring about an increased disease resistance (EPA 309862,        EPA0464461),    -   genetically modified plants with reduced photorespiration which        feature higher yields and higher stress tolerance (EPA 0305398),    -   transgenic crop plants which produce pharmaceutically or        diagnostically important proteins (“molecular pharming”),    -   transgenic crop plants which are distinguished by higher yields        or better quality,    -   transgenic crop plants which are distinguished by a combination        of novel properties such as a combination of the abovementioned        novel properties (“gene stacking”).

A large number of molecular-biological techniques by means of whichnovel transgenic plants with modified properties can be generated areknown in principle; see, for example, I. Potrykus and G. Spangenberg(eds.) Gene Transfer to Plants, Springer Lab Manual (1995), SpringerVerlag Berlin, Heidelberg, or Christou, “Trends in Plant Science” 1(1996) 423-431).

To carry out such recombinant manipulations, it is possible to introducenucleic acid molecules into plasmids, which permit a mutagenesis orsequence modification by recombination of DNA sequences. For example,base substitutions can be carried out, part-sequences can be removed, ornatural or synthetic sequences may be added with the aid of standardmethods. To link the DNA fragments with one another, it is possible toadd adapters or linkers to the fragments; see, for example, Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, 2. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; or Winnacker “Geneand Klone”, VCH Weinheim 2. ed., 1996

The generation of plant cells with a reduced activity for a gene productcan be achieved for example by the expression of at least onecorresponding antisense RNA, a sense RNA for achieving a cosuppressioneffect, or a combination of both an antisense and sense RNA forming adouble-stranded silencing RNA molecule (RNAi), or by the expression ofat least one correspondingly constructed ribozyme, which specificallycleaves transcripts of the abovementioned gene product. To do this, itis possible firstly to use DNA molecules which comprise all of thecoding sequence of a gene product, including any flanking sequenceswhich may be present, or else DNA molecules which only comprise parts ofthe coding sequence, it being necessary for these parts to be longenough to bring about an antisense effect in the cells. It is alsopossible to use DNA sequences which have a high degree of homology withthe coding sequences of a gene product, but which are not entirelyidentical.

When expressing nucleic acid molecules in plants, the obtained proteinmay be localized in any compartment of the plant cell. In order toachieve localization in a particular compartment, however, it ispossible for example to link the coding region to DNA sequences whichensure the localization in a specific compartment. Such sequences areknown to the skilled person (see, for example, Braun et al., EMBO J. 11(1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988),846-850; Sonnewald et al., Plant J. 1 (1991), 95-106). However, thenucleic acid molecules can also be expressed in the organelles of theplant cells.

The transgenic plant cells can be regenerated by known techniques togive intact plants. In principle, the transgenic plants may be plants ofany plant species, including monocotyledonous or dicotyledonous plants.

Thus, transgenic plants can be obtained which—in addition to thechimeric HPPD gene of the invention—have modified properties as theresult of overexpression, suppression or inhibition of homologous(=natural) genes or gene sequences or expression of heterologous(=foreign) genes or gene sequences.

On the plants, plant cells or seeds of the invention, it is preferred toemploy the HPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione intransgenic crops which are also resistant to growth regulators such as,for example, 2,4-D or dicamba, or against herbicides which inhibitessential plant enzymes, for example acetolactate synthases (ALS), EPSPsynthases, or glutamine synthases (GS), or against herbicides from thegroup of the sulfonylureas, glyphosate, or glufosinate and analogousactive substances.

The invention therefore also relates to the use of herbicides applied tothis HPPD tolerant plants according to the invention for controllingharmful plants (i.e. weeds) which also extends to transgenic crop plantscomprising a second or more herbicide resistance(s) beside theresistance against HPPD inhibitor herbicide of the class of triketones,such as tembotrione, sulcotrione and mesotrione, of the class ofisoxazoles such as isoxaflutole or of the class of pyrazolinates, suchas pyrasulfotole and topramezone, particularly selected fromtembotrione, sulcotrione, topramezone, bicyclopyrone, tefuryltrione andmesotrione, more particularly tembotrione.

HPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione can beemployed in the customary preparations in the form of wettable powders,emulsifiable concentrates, sprayable solutions, dusts or granules.

HPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione can beformulated in various ways, depending on the prevailing biologicaland/or physico-chemical parameters. Examples of possible formulationsare: wettable powders (WP), water-soluble powders (SP), water-solubleconcentrates, emulsifiable concentrates (EC), emulsions (EW), such asoil-in-water and water-in-oil emulsions, sprayable solutions, suspensionconcentrates (SC), oil- or water-based dispersions, oil-misciblesolutions, capsule suspensions (CS), dusts (DP), seed-dressing products,granules for application by broadcasting and on the soil, granules (GR)in the form of microgranules, spray granules, coated granules andadsorption granules, water-dispersible granules (WG), water-solublegranules (SG), ULV formulations, microcapsules and waxes.

These individual types of formulation are known in principle and aredescribed, for example, in: Winnacker-Kuchler, “Chemische Technologie”[Chemical technology], volume 7, C. Hanser Verlag Munich, 4th Ed. 1986;Wade van Valkenburg, “Pesticide Formulations”, Marcel Dekker, N.Y.,1973; K. Martens, “Spray Drying” Handbook, 3rd Ed. 1979, G. Goodwin Ltd.London.

The formulation auxiliaries required, such as inert materials,surfactants, solvents and further additives, are also known and aredescribed, for example, in: Watkins, “Handbook of Insecticide DustDiluents and Carriers”, 2nd Ed., Darland Books, Caldwell N.J., H. v.Olphen, “Introduction to Clay Colloid Chemistry”; 2nd Ed., J. Wiley &Sons, N.Y.; C. Marsden, “Solvents Guide”; 2nd Ed., Interscience, N.Y.1963; McCutcheon's “Detergents and Emulsifiers Annual”, MC Publ. Corp.,Ridgewood N.J.; Sisley and Wood, “Encyclopedia of Surface ActiveAgents”, Chem. Publ. Co. Inc., N.Y. 1964; Schönfeldt,“Grenzflächenaktive Äthylenoxidaddukte” [Interface-active ethylene oxideadducts], Wiss. Verlagsgesell., Stuttgart 1976; Winnacker-Küchler,“Chemische Technologie” [Chemical technology], volume 7, C. HanserVerlag Munich, 4th Ed. 1986.

Based on these formulations, it is also possible to prepare combinationswith other pesticidally active substances such as, for example,insecticides, acaricides, herbicides, fungicides, and with safeners,fertilizers and/or growth regulators, for example in the form of a readymix or a tank mix.

Wettable powders are preparations which are uniformly dispersible inwater and which, besides the active substance, also comprise ionicand/or nonionic surfactants (wetters, dispersers), for examplepolyoxyethylated alkylphenols, polyoxyethylated fatty alcohols,polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates,alkanesulfonates, alkylbenzenesulfonates, sodium lignosulfonate, sodium2,2′-dinaphthylmethane-6,6′-disulfonate, sodiumdibutylnaphthalenesulfonate or else sodium oleoylmethyltaurinate,besides a diluent or inert substance. To prepare the wettable powders,the herbicidally active substances are ground finely, for example incustomary apparatuses such as hammer mills, blower mills and air-jetmills, and mixed with the formulation auxiliaries, either simultaneouslyor subsequently.

Emulsifiable concentrates are prepared by dissolving the activesubstance in an organic solvent, for example butanol, cyclohexanone,dimethylformamide, xylene or else higher-boiling aromatics orhydrocarbons or mixtures of the organic solvents with addition of one ormore ionic and/or nonionic surfactants (emulsifiers). Examples ofemulsifiers which may be used are: calcium alkylarylsulfonates such ascalcium dodecylbenzenesulfonate, or nonionic emulsifiers such as fattyacid polyglycol esters, alkylarylpolyglycol ethers, fatty alcoholpolyglycol ethers, propylene oxide/ethylene oxide condensates, alkylpolyethers, sorbitan esters such as, for example, sorbitan fatty acidesters or polyoxyethylene sorbitan esters such as, for example,polyoxyethylene sorbitan fatty acid esters.

Dusts are obtained by grinding the active substance with finely dividedsolid materials such as, for example, talcum, natural clays such askaolin, bentonite and pyrophyllite, or diatomaceous earth.

Suspension concentrates can be water- or oil-based. They can be preparedfor example by wet-grinding by means of commercially available beadmills, if appropriate with addition of surfactants as already listedabove for example in the case of the other formulation types.

Emulsions, for example oil-in-water emulsions (EW), can be prepared forexample by means of stirrers, colloid mills and/or static mixers usingaqueous organic solvents and, if appropriate, surfactants, as havealready been mentioned for example above for the other formulationtypes.

Granules can be prepared either by spraying the active substance ontoadsorptive, granulated inert material, or by applying active substanceconcentrates to the surface of carriers such as sand, kaolinites orgranulated inert material with the aid of stickers, for examplepolyvinyl alcohol, sodium polyacrylate or else mineral oils. Suitableactive substances can also be granulated in the manner which iscustomary for the production of fertilizer granules, if desired as amixture with fertilizers.

Water-dispersible granules are generally prepared by customary methodssuch as spray drying, fluidized-bed granulation, disk granulation,mixing with high-speed stirrers, and extrusion without solid inertmaterial.

To prepare disk granules, fluidized-bed granules, extruder granules andspray granules, see, for example, methods in “Spray-Drying Handbook” 3rded. 1979, G. Goodwin Ltd., London; J. E. Browning, “Agglomeration”,Chemical and Engineering 1967, pages 147 et seq.; “Perry's ChemicalEngineer's Handbook”, 5th Ed., McGraw-Hill, New York 1973, p. 8-57.

For further details of the formulation of crop protection products see,for example, G. C. Klingman, “Weed Control as a Science”, John Wiley andSons, Inc., New York, 1961, pages 81-96 and J. D. Freyer, S. A. Evans,“Weed Control Handbook”, 5th Ed., Blackwell Scientific Publications,Oxford, 1968, pages 101-103.

As a rule, the agrochemical preparations comprise from 0.1 to 99% byweight, in particular from 0.1 to 95% by weight, of compounds accordingto the invention. In wettable powders, the active substanceconcentration is, for example, approximately 10 to 90% by weight, theremainder to 100% by weight being composed of customary formulationconstituents. In the case of emulsifiable concentrates, the activesubstance concentration can amount to approximately 1 to 90, preferably5 to 80% by weight. Formulations in the form of dusts comprise from 1 to30% by weight of active substance, preferably in most cases from 5 to20% by weight of active substance, and sprayable solutions compriseapproximately from 0.05 to 80, preferably from 2 to 50% by weight ofactive substance. In the case of water-dispersible granules, the activesubstance content depends partly on whether the active compound is inliquid or solid form, and on the granulation auxiliaries, fillers andthe like which are being used. In the case of the water-dispersiblegranules, for example, the active substance content is between 1 and 95%by weight, preferably between 10 and 80% by weight.

In addition, the active substance formulations mentioned comprise, ifappropriate, the auxiliaries which are conventional in each case, suchas stickers, wetters, dispersants, emulsifiers, penetrations,preservatives, antifreeze agents, solvents, fillers, carriers,colorants, antifoams, evaporation inhibitors, and pH and viscosityregulators.

Based on these formulations, it is also possible to prepare combinationsof an HPPD inhibitor herbicide of the class of triketones, such astembotrione, sulcotrione and mesotrione, or of the class ofpyrazolinates, such as pyrasulfotole and topramezone, particularlyselected from tembotrione, sulcotrione, topramezone, bicyclopyrone,tefuryltrione and mesotrione, more particularly tembotrione with otherpesticidally active substances such as, for example, insecticides,acaricides, herbicides, fungicides, and with safeners, fertilizersand/or growth regulators, for example in the form of a ready mix or atank mix to be applied to HPPD tolerant plants according to theinvention.

Active substances which can be applied to HPPD tolerant plants accordingto the present invention in combination with HPPD inhibitor herbicide ofthe class of triketones, such as tembotrione, sulcotrione andmesotrione, or of the class of pyrazolinates, such as pyrasulfotole andtopramezone, particularly selected from tembotrione, sulcotrione,topramezone, bicyclopyrone, tefuryltrione and mesotrione, moreparticularly tembotrione in mixed formulations or in the tank mix are,for example, known active substances which are based on the inhibitionof, for example, acetolactate synthase, acetyl-CoA carboxylase,cellulose synthase, enolpyruvylshikimate-3-phosphate synthase, glutaminesynthetase, p-hydroxyphenylpyruvate dioxygenase, phytoene desaturase,photosystem I, photosystem II, protoporphyrinogen oxidase, as aredescribed in, for example, Weed Research 26 (1986) 441-445 or “ThePesticide Manual”, 14th edition, The British Crop Protection Council andthe Royal Soc. of Chemistry, 2003 and the literature cited therein.Known herbicides or plant growth regulators which can be combined withthe compounds according to the invention are, for example, the followingactive substances (the compounds are either designated by the commonname according to the International Organization for Standardization(ISO) or by a chemical name, if appropriate together with the codenumber) and always comprise all use forms such as acids, salts, estersand isomers such as stereoisomers and optical isomers. In this context,one and in some cases also several use forms are mentioned by way ofexample:

acetochlor, acibenzolar, acibenzolar-S-methyl, acifluorfen,acifluorfen-sodium, aclonifen, alachlor, allidochlor, alloxydim,alloxydim-sodium, ametryne, amicarbazone, amidochlor, amidosulfuron,aminocyclopyrachlor, aminopyralid, amitrole, ammonium sulfamate,ancymidol, anilofos, asulam, atrazine, azafenidin, azimsulfuron,aziprotryne, BAH-043, BAS-140H, BAS-693H, BAS-714H, BAS-762H, BAS-776H,BAS-800H, beflubutamid, benazolin, benazolin-ethyl, bencarbazone,benfluralin, benfuresate, bensulide, bensulfuron-methyl, bentazone,benzfendizone, benzobicyclon, benzofenap, benzofluor, benzoylprop,bifenox, bilanafos, bilanafos-sodium, bispyribac, bispyribac-sodium,bromacil, bromobutide, bromofenoxim, bromoxynil, bromuron, buminafos,busoxinone, butachlor, butafenacil, butamifos, butenachlor, butralin,butroxydim, butylate, cafenstrole, carbetamide, carfentrazone,carfentrazone-ethyl, chlomethoxyfen, chloramben, chlorazifop,chlorazifop-butyl, chlorbromuron, chlorbufam, chlorfenac,chlorfenac-sodium, chlorfenprop, chlorflurenol, chlorflurenol-methyl,chloridazon, chlorimuron, chlorimuron-ethyl, chlormequat-chloride,chlornitrofen, chlorophthalim, chlorthal-dimethyl, chlorotoluron,chlorsulfuron, cinidon, cinidon-ethyl, cinmethylin, cinosulfuron,clethodim, clodinafop clodinafop-propargyl, clofencet, clomazone,clomeprop, cloprop, clopyralid, cloransulam, cloransulam-methyl,cumyluron, cyanamide, cyanazine, cyclanilide, cycloate, cyclosulfamuron,cycloxydim, cycluron, cyhalofop, cyhalofop-butyl, cyperquat, cyprazine,cyprazole, 2,4-D, 2,4-DB, daimuron/dymron, dalapon, daminozide, dazomet,n-decanol, desmedipham, desmetryn, detosyl-pyrazolate (DTP), di-allate,dicamba, dichlobenil, dichlorprop, dichlorprop-P, diclofop,diclofop-methyl, diclofop-P-methyl, diclosulam, diethatyl,diethatyl-ethyl, difenoxuron, difenzoquat, diflufenican, diflufenzopyr,diflufenzopyr-sodium, dimefuron, dikegulac-sodium, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimetrasulfuron, dinitramine, dinoseb, dinoterb, diphenamid,dipropetryn, diquat, diquat-dibromide, dithiopyr, diuron, DNOC,eglinazine-ethyl, endothal, EPTC, esprocarb, ethalfluralin,ethametsulfuron-methyl, ethephon, ethidimuron, ethiozin, ethofumesate,ethoxyfen, ethoxyfen-ethyl, ethoxysulfuron, etobenzanid, F-5331, i.e.N-[2-chloro-4-fluoro-5-[4-(3-fluoro-propyl)-4,5-dihydro-5-oxo-1H-tetrazol-1-yl]-phenyl]ethanesulfonamide,fenoprop, fenoxaprop, fenoxaprop-P, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, flamprop,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop, fluazifop-P, fluazifop-butyl, fluazifop-P-butyl, fluazolate,flucarbazone, flucarbazone-sodium, flucetosulfuron, fluchloralin,flufenacet (thiafluamide), flufenpyr, flufenpyr-ethyl, flumetralin,flumetsulam, flumiclorac, flumiclorac-pentyl, flumioxazin, flumipropyn,fluometuron, fluorodifen, fluoroglycofen, fluoroglycofen-ethyl,flupoxam, flupropacil, flupropanate, flupyrsulfuron,flupyrsulfuron-methyl-sodium, flurenol, flurenol-butyl, fluridone,flurochloridone, fluroxypyr, fluroxypyr-meptyl, flurprimidol,flurtamone, fluthiacet, fluthiacet-methyl, fluthiamide, fomesafen,foramsulfuron, forchlorfenuron, fosamine, furyloxyfen, gibberellic acid,glufosinate, L-glufosinate, L-glufosinate-ammonium,glufosinate-ammonium, glyphosate, glyphosate-isopropylammonium, H-9201,halosafen, halosulfuron, halosulfuron-methyl, haloxyfop, haloxyfop-P,haloxyfop-ethoxyethyl, haloxyfop-P-ethoxyethyl, haloxyfop-methyl,haloxyfop-P-methyl, hexazinone, HNPC-9908, HOK-201, HW-02,imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, imazapyr,imazaquin, imazethapyr, imazosulfuron, inabenfide, indanofan,indoleacetic acid (IAA), 4-indol-3-ylbutyric acid (IBA), iodosulfuron,iodosulfuron-methyl-sodium, ioxynil, isocarbamid, isopropalin,isoproturon, isouron, isoxaben, isoxachlortole, isoxaflutole,isoxapyrifop, KUH-043, KUH-071, karbutilate, ketospiradox, lactofen,lenacil, linuron, maleic hydrazide, MCPA, MCPB, MCPB-methyl, -ethyl and-sodium, mecoprop, mecoprop-sodium, mecoprop-butotyl,mecoprop-P-butotyl, mecoprop-P-dimethylammonium,mecoprop-P-2-ethylhexyl, mecoprop-P-potassium, mefenacet, mefluidide,mepiquat-chloride, mesosulfuron, mesosulfuron-methyl,methabenzthiazuron, metam, metamifop, metamitron, metazachlor,methazole, methoxyphenone, methyldymron, 1-methylcyclopropene, methylisothiocyanate, metobenzuron, metobenzuron, metobromuron, metolachlor,S-metolachlor, metosulam, metoxuron, metribuzin, metsulfuron,metsulfuron-methyl, molinate, monalide, monocarbamide, monocarbamidedihydrogen sulfate, monolinuron, monosulfuron, monuron, MT 128, MT-5950,i.e. N-[3-chloro-4-(1-methylethyl)-phenyl]-2-methylpentanamide,NGGC-011, naproanilide, napropamide, naptalam, NC-310, i.e.4-(2,4-dichlorobenzoyl)-1-methyl-5-benzyloxypyrazole, neburon,nicosulfuron, nipyraclofen, nitralin, nitrofen, nitrophenolat-sodium(isomer mixture), nitrofluorfen, nonanoic acid, norflurazon, orbencarb,orthosulfamuron, oryzalin, oxadiargyl, oxadiazon, oxasulfuron,oxaziclomefone, oxyfluorfen, paclobutrazole, paraquat, paraquatdichloride, pelargonic acid (nonanoic acid), pendimethalin, pendralin,penoxsulam, pentanochlor, pentoxazone, perfluidone, pethoxamid,phenisopham, phenmedipham, phenmedipham-ethyl, picloram, picolinafen,pinoxaden, piperophos, pirifenop, pirifenop-butyl, pretilachlor,primisulfuron, primisulfuron-methyl, probenazole, profluazol,procyazine, prodiamine, prifluraline, profoxydim, prohexadione,prohexadione-calcium, prohydrojasmone, prometon, prometryn, propachlor,propanil, propaquizafop, propazine, propham, propisochlor,propoxycarbazone, propoxycarbazone-sodium, propyzamide, prosulfalin,prosulfocarb, prosulfuron, prynachlor, pyraclonil, pyraflufen,pyraflufen-ethyl, pyrazolynate (pyrazolate), pyrazosulfuron-ethyl,pyrazoxyfen, pyribambenz, pyribambenz-isopropyl, pyribenzoxim,pyributicarb, pyridafol, pyridate, pyriftalid, pyriminobac,pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyrithiobac-sodium,pyroxasulfone, pyroxsulam, quinclorac, quinmerac, quinoclamine,quizalofop, quizalofop-ethyl, quizalofop-P, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, saflufenacil, secbumeton, sethoxydim,siduron, simazine, simetryn, SN-106279, sulf-allate (CDEC),sulfentrazone, sulfometuron, sulfometuron-methyl, sulfosate(glyphosate-trimesium), sulfosulfuron, SYN-523, SYP-249, SYP-298,SYP-300, tebutam, tebuthiuron, tecnazene, tepraloxydim, terbacil,terbucarb, terbuchlor, terbumeton, terbuthylazine, terbutryne, TH-547,thenylchlor, thiafluamide, thiazafluron, thiazopyr, thidiazimin,thidiazuron, thiencarbazone, thiencarbazone-methyl, thifensulfuron,thifensulfuron-methyl, thiobencarb, tiocarbazil, tralkoxydim,tri-allate, triasulfuron, triaziflam, triazofenamide, tribenuron,tribenuron-methyl, trichloroacetic acid (TCA), triclopyr, tridiphane,trietazine, trifloxysulfuron, trifloxysulfuron-sodium, trifluralin,triflusulfuron, triflusulfuron-methyl, trimeturon, trinexapac,trinexapac-ethyl, tritosulfuron, tsitodef, uniconazole, uniconazole-P,vernolate, ZJ-0166, ZJ-0270, ZJ-0543, ZJ-0862 and the followingcompounds

The application rate required of the HPPD inhibitor herbicide of theclass of triketones, such as tembotrione, sulcotrione and mesotrione, orof the class of pyrazolinates, such as pyrasulfotole and topramezone,particularly selected from tembotrione, sulcotrione, topramezone,bicyclopyrone, tefuryltrione and mesotrione, more particularlytembotrione to be applied to areas where HPPD tolerant plants accordingto the present invention are growing varies as a function of theexternal conditions such as temperature, humidity, the nature of theherbicide used and the like. It can vary within wide limits, for examplebetween 0.001 and 1.0 kg/ha and more of active substance, but it ispreferably between 0.005 and 750 g/ha.

In case of combined applications of HPPD inhibitor herbicides withherbicides that differ from HPPD inhibitor herbicides to the HPPDtolerant plants according to the present invention, these mixtures maycause crop injury, based on the presence of the non HPPD inhibitorherbicides. In order to reduce/eliminate such crop injuries, appropriatesafeners may be added. These safeners, which are employed inantidotically active amounts, reduce the phytotoxic side effects ofherbicides/pesticides used, for example in economically important crops,such as cereals (wheat, barley, rye, corn, rice, millet), alfalfa, sugarbeet, sugarcane, oilseed rape, cotton and soya spp., preferably corn,cotton, sugarbeet, or soya spp.

The safeners are preferably selected from the group consisting of:

A) compounds of the formula (S-I)

where the symbols and indices have the following meanings:

-   -   n_(A) is a natural number from 0 to 5, preferably from 0 to 3;    -   R_(A) ¹ is halogen, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, nitro or        (C₁-C₄)-haloalkyl;    -   W_(A) is an unsubstituted or substituted divalent heterocyclic        radical from the group consisting of partially unsaturated or        aromatic five-membered heterocycles having 1 to 3 hetero ring        atoms of the type N or O, where at least one nitrogen atom and        at most one oxygen atom is present in the ring, preferably a        radical from the group consisting of (W_(A) ¹) to (W_(A) ⁴),

-   -   m_(A) is 0 or 1;    -   R_(A) ² is OR_(A) ³, SR_(A) ³ or NR_(A) ³R_(A) ⁴ or a saturated        -   or unsaturated 3- to 7-membered heterocycle having at least            one nitrogen atom and up to 3 heteroatoms, preferably from            the group consisting of O and S, which is attached via the            nitrogen atom to the carbonyl group in (S-I) and which is            unsubstituted or substituted by radicals from the group            consisting of (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy and optionally            substituted phenyl, preferably a radical of the formula            OR_(A) ³, NHR_(A) ⁴ or N(CH₃)₂, in particular of the formula            OR_(A) ³;    -   R_(A) ³ is hydrogen or an unsubstituted or substituted aliphatic        hydrocarbon radical having preferably a total of 1 to 18 carbon        atoms;    -   R_(A) ⁴ is hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkoxy or        substituted or unsubstituted phenyl;    -   R_(A) ⁵ is H, (C₁-C₈)-alkyl, (C₁-C₈)-haloalkyl),        (C₁-C₄)-alkoxy-(C₁-C₈)-alkyl, cyano or COOR_(A) ⁹ where R_(A) ⁹        is hydrogen, (C₁-C₈)-alkyl, (C₁-C₈)-haloalkyl,        (C₁-C₄)-alkoxy-(C₁-C₄)-alkyl, (C₁-C₆)-hydroxyalkyl,        (C₃-C₁₂)-cycloalkyl or tri-(C₁-C₄)-alkylsilyl;    -   R_(A) ⁶, R_(A) ⁷, R_(A) ⁸ are identical or different and are        hydrogen, (C₁-C₈)-alkyl, (C₁-C₈)-haloalkyl, (C₃-C₁₂)-cycloalkyl        or substituted or unsubstituted phenyl;

preferably:

a) compounds of the type of the dichlorophenylpyrazoline-3-carboxylicacid, preferably compounds such as ethyl1-(2,4-dichlorophenyl)-5-(ethoxycarbonyl)-5-methyl-2-pyrazoline-3-carboxylate(S1-1) (“mefenpyr-diethyl”, see Pestic. Man.), and related compounds, asdescribed in WO 91/07874;

b) derivatives of dichlorophenylpyrazolecarboxylic acid, preferablycompounds such as ethyl1-(2,4-dichlorophenyl)-5-methylpyrazole-3-carboxylate (S1-2), ethyl1-(2,4-dichlorophenyl)-5-isopropylpyrazole-3-carboxylate (S1-3), ethyl1-(2,4-dichlorophenyl)-5-(1,1-dimethylethyl)pyrazole-3-carboxylate(S1-4), ethyl 1-(2,4-dichlorophenyl)-5-phenylpyrazole-3-carboxylate(S1-5) and related compounds, as described in EP-A-333 131 and EP-A-269806;

c) compounds of the type of the triazolecarboxylic acids, preferablycompounds such as fenchlorazole(-ethyl ester), i.e. ethyl1-(2,4-dichlorophenyl)-5-trichloro-methyl-(1H)-1,2,4-triazole-3-carboxylate(S1-6), and related compounds, as described in EP-A-174 562 and EP-A-346620;

d) compounds of the type of the 5-benzyl- or5-phenyl-2-isoxazoline-3-carboxylic acid or the5,5-diphenyl-2-isoxazoline-3-carboxylic acid, preferably compounds suchas ethyl 5-(2,4-dichlorobenzyl)-2-isoxazoline-3-carboxylate (S1-7) orethyl 5-phenyl-2-isoxazoline-3-carboxylate (S1-8) and related compounds,as described in WO 91/08202, or ethyl5,5-diphenyl-2-isoxazolinecarboxylate (S1-9) (“isoxadifen-ethyl”) orn-propyl 5,5-diphenyl-2-isoxazolinecarboxylate (S1-10) or ethyl5-(4-fluorophenyl)-5-phenyl-2-isoxazoline-3-carboxylate (S1-11), asdescribed in the patent application WO-A-95/07897.

B) Quinoline derivatives of the formula (S-II)

where the symbols and indices have the following meanings:

R_(B) ¹ is halogen, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, nitro or(C₁-C₄)-haloalkyl;

n_(B) is a natural number from 0 to 5, preferably from 0 to 3;

R_(B) ² OR_(B) ³, SR_(B) ³ or NR_(B) ³R_(B) ⁴ or a saturated

or unsaturated 3- to 7-membered heterocycle having at least one nitrogenatom and up to 3 heteroatoms, preferably from the group consisting of Oand S, which is attached via the nitrogen atom to the carbonyl group in(S-II) and is unsubstituted or substituted by radicals from the groupconsisting of (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy or optionally substitutedphenyl, preferably a radical of the formula OR_(B) ³, NHR_(B) ⁴ orN(CH₃)₂, in particular of the formula OR_(B) ³;

R_(B) ³ is hydrogen or an unsubstituted or substituted aliphatichydrocarbon radical having preferably a total of 1 to 18 carbon atoms;

R_(B) ⁴ is hydrogen, (C₁-C₆)-alkyl, (C₁-C₆)-alkoxy or substituted orunsubstituted phenyl;

T_(B) is a (C₁- or C₂)-alkanediyl chain which is unsubstituted orsubstituted by one or two (C₁-C₄)-alkyl radicals or by[(C₁-C₃)-alkoxy]carbonyl;

preferably:

a) compounds of the type of the 8-quinolinoxyacetic acid (S2),preferably

-   1-methylhexyl (5-chloro-8-quinolinoxy)acetate (common name    “cloquintocet-mexyl” (S2-1) (see Pestic. Man.),-   1,3-dimethylbut-1-yl (5-chloro-8-quinolinoxy)acetate (S2-2),-   4-allyloxybutyl (5-chloro-8-quinolinoxy)acetate (S2-3),-   1-allyloxyprop-2-yl (5-chloro-8-quinolinoxy)acetate-(S2-4),-   ethyl (5-chloro-8-quinolinoxy)acetate (S2-5),-   methyl (5-chloro-8-quinolinoxy)acetate (S2-6),-   allyl (5-chloro-8-quinolinoxy)acetate (S2-7),-   2-(2-propylideneiminoxy)-1-ethyl (5-chloro-8-quinolinoxy)acetate    (S2-8), 2-oxoprop-1-yl (5-chloro-8-quinolinoxy)acetate (S2-9) and    related compounds, as described in EP-A-86 750, EP-A-94 349 and    EP-A-191 736 or EP-A-0 492 366, and also their hydrates and salts,    as described in WO-A-2002/034048.

b) Compounds of the type of the (5-chloro-8-quinolinoxy)malonic acid,preferably compounds such as diethyl (5-chloro-8-quinolinoxy)malonate,diallyl (5-chloro-8-quinolinoxy)malonate, methyl ethyl(5-chloro-8-quinolinoxy)malonate and related compounds, as described inEP-A-0 582 198.

C) Compounds of the formula (S-III)

where the symbols and indices have the following meanings:

R_(C) ¹ is (C₁-C₄)-alkyl, (C₁-C₄)-haloalkyl, (C₂-C₄)-alkenyl,(C₂-C₄)-haloalkenyl, (C₃-C₇)-cycloalkyl, preferably dichloromethyl;

R_(C) ², R_(C) ³ are identical or different and are hydrogen,(C₁-C₄)-alkyl, (C₂-C₄)-alkenyl, (C₂-C₄)-alkynyl, (C₁-C₄)-haloalkyl,(C₂-C₄)-haloalkenyl, (C₁-C₄)-alkylcarbamoyl-(C₁-C₄)-alkyl,(C₂-C₄)-alkenylcarbamoyl-(C₁-C₄)-alkyl, (C₁-C₄)-alkoxy-(C₁-C₄)-alkyl,dioxolanyl-(C₁-C₄)-alkyl, thiazolyl, furyl, furylalkyl, thienyl,piperidyl, substituted or unsubstituted phenyl, or R_(C) ² and R_(C) ³together form a substituted or unsubstituted heterocyclic ring,

preferably an oxazolidine, thiazolidine, piperidine, morpholine,hexahydropyrimidine or benzoxazine ring;

preferably:

active compounds of the type of the dichloroacetamides which arefrequently used as pre-emergence safener (soil-acting safeners), suchas, for example,

“dichlormid” (see Pestic.Man.) (=N,N-diallyl-2,2-dichloroacetamide),

“R-29148” (=3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidine fromStauffer),

“R-28725” (=3-dichloroacetyl-2,2,-dimethyl-1,3-oxazolidine fromStauffer),

“benoxacor” (see Pestic. Man.)(=4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzoxazine),

“PPG-1292” (=N-allyl-N-[(1,3-dioxolan-2-yl)methyl]dichloroacetamide fromPPG Industries),

“DKA-24” (=N-allyl-N-[(allylaminocarbonyl)methyl]dichloroacetamide fromSagro-Chem),

“AD-67” or “MON 4660” (=3-dichloroacetyl-1-oxa-3-aza-spiro[4,5]decanefrom Nitrokemia or Monsanto),

“TI-35” (=1-dichloroacetylazepane from TRI-Chemical RT)

“diclonon” (dicyclonone) or “BAS145138” or “LAB145138”(=3-dichloroacetyl-2,5,5-trimethyl-1,3-diazabicyclo[4.3.0]nonane fromBASF) and

“furilazole” or “MON 13900” (see Pestic. Man.)(=(RS)-3-dichloroacetyl-5-(2-furyl)-2,2-dimethyloxazolidine).

D) N-Acylsulfonamides of the formula (S-IV) and their salts

in which

X_(D) is CH or N;

R_(D) ¹ is CO—NR_(D) ⁵R_(D) ⁶ or NHCO—R_(D) ⁷;

R_(D) ² is halogen, (C₁-C₄)-haloalkyl, (C₁-C₄)-haloalkoxy, nitro,(C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, (C₁-C₄)-alkylsulfonyl,(C₁-C₄)-alkoxycarbonyl or (C₁-C₄)-alkylcarbonyl;

R_(D) ³ is hydrogen, (C₁-C₄)-alkyl, (C₂-C₄)-alkenyl or (C₂-C₄)-alkynyl;

R_(D) ⁴ is halogen, nitro, (C₁-C₄)-alkyl, (C₁-C₄)-haloalkyl,(C₁-C₄)-haloalkoxy, (C₃-C₆)-cycloalkyl, phenyl, (C₁-C₄)-alkoxy, cyano,(C₁-C₄)-alkylthio, (C₁-C₄)-alkylsulfinyl, (C₁-C₄)-alkylsulfonyl,(C₁-C₄)-alkoxycarbonyl or (C₁-C₄)-alkylcarbonyl;

R_(D) ⁵ is hydrogen, (C₁-C₆)-alkyl, (C₃-C₆)-cycloalkyl, (C₂-C₆)-alkenyl,(C₂-C₆)-alkynyl, (C₅-C₆)-cycloalkenyl, phenyl or 3- to 6-memberedheterocyclyl containing v_(D) heteroatoms from the group consisting ofnitrogen, oxygen and sulfur, where the seven last-mentioned radicals aresubstituted by v_(D) substituents from the group consisting of halogen,(C₁-C₆)-alkoxy, (C₁-C₆)-haloalkoxy, (C₁-C₂)-alkylsulfinyl,(C₁-C₂)-alkylsulfonyl, (C₃-C₆)-cycloalkyl, (C₁-C₄)-alkoxycarbonyl,(C₁-C₄)-alkylcarbonyl and phenyl and, in the case of cyclic radicals,also (C₁-C₄)-alkyl and (C₁-C₄)-haloalkyl;

R_(D) ⁶ is hydrogen, (C₁-C₆)-alkyl, (C₂-C₆)-alkenyl or (C₂-C₆)-alkynyl,where the three last-mentioned radicals are substituted by v_(D)radicals from the group consisting of halogen, hydroxy, (C₁-C₄)-alkyl,(C₁-C₄)-alkoxy and (C₁-C₄)-alkylthio, or

R_(D) ⁵ and R_(D) ⁶ together with the nitrogen atom carrying them form apyrrolidinyl or piperidinyl radical;

R_(D) ⁷ is hydrogen, (C₁-C₄)-alkylamino, di-(C₁-C₄)-alkylamino,(C₁-C₆)-alkyl, (C₃-C₆)-cycloalkyl, where the 2 last-mentioned radicalsare substituted by v_(D) substituents from the group consisting ofhalogen, (C₁-C₄)-alkoxy, halogen-(C₁-C₆)-alkoxy and (C₁-C₄)-alkylthioand, in the case of cyclic radicals, also (C₁-C₄)-alkyl and(C₁-C₄)-haloalkyl;

n_(D) is 0, 1 or 2;

m_(D) is 1 or 2;

v_(D) is 0, 1, 2 or 3;

from among these, preference is given to compounds of the type of theN-acylsulfonamides, for example of the formula (S-V) below, which areknown, for example, from WO 97/45016

in which

R_(D) ⁷ is (C₁-C₆)-alkyl, (C₃-C₆)-cycloalkyl, where the 2 last-mentionedradicals are substituted by v_(D) substituents from the group consistingof halogen, (C₁-C₄)-alkoxy, halogen-(C₁-C₆)-alkoxy and (C₁-C₄)-alkylthioand, in the case of cyclic radicals, also (C₁-C₄)-alkyl and(C₁-C₄)-haloalkyl;

R_(D) ⁴ is halogen, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, CF₃;

m_(D) is 1 or 2;

v_(D) is 0, 1, 2 or 3;

and also

acylsulfamoylbenzamides, for example of the formula (S-VI) below, whichare known, for example, from WO 99/16744,

for example those in which

R_(D) ⁵=cyclopropyl and (R_(D) ⁴)=2-OMe (“cyprosulfamide”, S3-1),

R_(D) ⁵=cyclopropyl and (R_(D) ⁴)=5-Cl-2-OMe (S3-2),

R_(D) ⁵=ethyl and (R_(D) ⁴)=2-OMe (S3-3),

R_(D) ⁵=isopropyl and (R_(D) ⁴)=5-Cl-2-OMe (S3-4) and

R_(D) ⁵=isopropyl and (R_(D) ⁴)=2-OMe (S3-5);

and also

compounds of the type of the N-acylsulfamoylphenylureas of the formula(S-VII), which are known, for example, from EP-A-365484

in which

R_(D) ⁸ and R_(D) ⁹ independently of one another are hydrogen,(C₁-C₈)-alkyl, (C₃-C₈)-cycloalkyl, (C₃-C₆)-alkenyl, (C₃-C₆)-alkynyl,

R_(D) ⁴ is halogen, (C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, CF₃

m_(D) is 1 or 2;

from among these in particular

-   1-[4-(N-2-methoxybenzoylsulfamoyl)phenyl]-3-methylurea,-   1-[4-(N-2-methoxybenzoylsulfamoyl)phenyl]-3,3-dimethylurea,-   1-[4-(N-4,5-dimethylbenzoylsulfamoyl)phenyl]-3-methylurea,-   1-[4-(N-naphthoylsulfamoyl)phenyl]-3,3-dimethylurea,

G) active compounds from the class of the hydroxyaromatics andaromatic-aliphatic carboxylic acid derivatives, for example

ethyl 3,4,5-triacetoxybenzoate, 3,5-dimethoxy-4-hydroxybenzoic acid,3,5-dihydroxybenzoic acid, 4-hydroxysalicylic acid, 4-fluorosalicyclicacid, 1,2-dihydro-2-oxo-6-trifluoromethylpyridine-3-carboxamide,2-hydroxycinnamic acid, 2,4-dichlorocinnamic acid, as described in WO2004084631, WO 2005015994, WO 2006007981, WO 2005016001;

H) active compounds from the class of the 1,2-dihydroquinoxalin-2-ones,for example

1-methyl-3-(2-thienyl)-1,2-dihydroquinoxalin-2-one,1-methyl-3-(2-thienyl)-1,2-dihydroquinoxaline-2-thione,1-(2-aminoethyl)-3-(2-thienyl)-1,2-dihydroquinoxalin-2-onehydrochloride,1-(2-methylsulfonylaminoethyl)-3-(2-thienyl)-1,2-dihydro-quinoxalin-2-one,as described in WO 2005112630,

I) active compounds which, in addition to a herbicidal action againstharmful plants, also have safener action on crop plants such as rice,such as, for example, “dimepiperate” or “MY-93” (see Pestic. Man.)(=S-1-methyl-1-phenylethyl piperidine-1-thiocarboxylate), which is knownas safener for rice against damage by the herbicide molinate,

“daimuron” or “SK 23” (see Pestic. Man.)(=1-(1-methyl-1-phenylethyl)-3-p-tolyl-urea), which is known as safenerfor rice against damage by the herbicide imazosulfuron,

“cumyluron”=“JC-940”(=3-(2-chlorophenylmethyl)-1-(1-methyl-1-phenyl-ethyl)urea, seeJP-A-60087254), which is known as safener for rice against damage by anumber of herbicides,

“methoxyphenone” or “NK 049” (=3,3′-dimethyl-4-methoxybenzophenone),which is known as safener for rice against damage by a number ofherbicides,

“CSB” (=1-bromo-4-(chloromethylsulfonyl)benzene) (CAS Reg. No.54091-06-4 from Kumiai), which is known as safener against damage by anumber of herbicides in rice,

K) compounds of the formula (S-IX),

-   -   as described in WO-A-1998/38856

in which the symbols and indices have the following meanings:

R_(K) ¹, R_(K) ² independently of one another are halogen,(C₁-C₄)-alkyl, (C₁-C₄)-alkoxy, (C₁-C₄)-haloalkyl, (C₁-C₄)-alkylamino,di-(C₁-C₄)-alkylamino, nitro;

A_(K) is COOR_(K) ³ or COOR_(K) ⁴

R_(K) ³, R_(K) ⁴ independently of one another are hydrogen,(C₁-C₄)-alkyl, (C₂-C₆)-alkenyl, (C₂-C₄)-alkynyl, cyanoalkyl,(C₁-C₄)-haloalkyl, phenyl, nitrophenyl, benzyl, halobenzyl,pyridinylalkyl or alkylammonium,

n_(K) ¹ is 0 or 1,

n_(K) ², n_(K) ³ independently of one another are 0, 1 or 2

preferably: methyl(diphenylmethoxy)acetate (CAS Reg. No.: 41858-19-9),

L) compounds of the formula (S-X),

-   -   as described in WO A-98/27049

in which the symbols and indices have the following meanings:

X_(L) is CH or N,

n_(L) is, in the case that X═N, an integer from 0 to 4 and,

-   -   in the case that X═CH, an integer from 0 to 5,    -   R_(L) ¹ is halogen, (C₁-C₄)-alkyl, (C₁-C₄)-haloalkyl,        (C₁-C₄)-alkoxy, (C₁-C₄)-haloalkoxy, nitro, (C₁-C₄)-alkylthio,        (C₁-C₄)-alkylsulfonyl, (C₁-C₄)-alkoxycarbonyl, optionally        substituted phenyl, optionally substituted phenoxy,

R_(L) ² is hydrogen or (C₁-C₄)-alkyl,

R_(L) ³ is hydrogen, (C₁-C₈)-alkyl, (C₂-C₄)-alkenyl, (C₂-C₄)-alkynyl oraryl, where each of the carbon-containing radicals mentioned above isunsubstituted or substituted by one or more, preferably by up to three,identical or different radicals from the group consisting of halogen andalkoxy; or salts thereof,

M) active compounds from the class of the3-(5-tetrazolylcarbonyl)-2-quinolones, for example

1,2-dihydro-4-hydroxy-1-ethyl-3-(5-tetrazolylcarbonyl)-2-quinolone (CASReg. No.: 219479-18-2),1,2-dihydro-4-hydroxy-1-methyl-3-(5-tetrazolylcarbonyl)-2-quinolone (CASReg. No.: 95855-00-8), as described in WO-A-1999000020,

N) compounds of the formula (S-XI) or (S-XII),

-   -   as described in WO-A-2007023719 and WO-A-2007023764

in which

R_(N) ¹ is halogen, (C₁-C₄)-alkyl, methoxy, nitro, cyano, CF₃, OCF₃

Y, Z independently of one another are O or S,

n_(N) is an integer from 0 to 4,

R_(N) ² is (C₁-C₁₆)-alkyl, (C₂-C₆)-alkenyl, (C₃-C₆)-cycloalkyl, aryl,benzyl, halobenzyl,

R_(N) ³ is hydrogen, (C₁-C₆)alkyl,

O) one or more compounds from the group consisting of:

-   1,8-naphthalic anhydride,-   O,O-diethyl S-2-ethylthioethyl phosphorodithioate (disulfoton),-   4-chlorophenyl methylcarbamate (mephenate),-   O,O-diethyl O-phenyl phosphorothioate (dietholate),-   4-carboxy-3,4-dihydro-2H-1-benzopyran-4-acetic acid (CL-304415, CAS    Reg. No.: 31541-57-8),-   2-propenyl 1-oxa-4-azaspiro[4.5]decane-4-carbodithioate (MG-838, CAS    Reg. No.: 133993-74-5),-   methyl [(3-oxo-1H-2-benzothiopyran-4(3H)-ylidene)methoxy]acetate    (from WO-A-98/13361; CAS Reg. No.: 205121-04-6),-   cyanomethoxyimino(phenyl)acetonitrile (cyometrinil),-   1,3-dioxolan-2-ylmethoxyimino(phenyl)acetonitrile (oxabetrinil),-   4′-chloro-2,2,2-trifluoroacetophenone O-1,3-dioxolan-2-ylmethyloxime    (fluxofenim),-   4,6-dichloro-2-phenylpyrimidine (fenclorim),-   benzyl 2-chloro-4-trifluoromethyl-1,3-thiazole-5-carboxylate    (flurazole),-   2-dichloromethyl-2-methyl-1,3-dioxolane (MG-191),

including the stereoisomers, and the salts customary in agriculture.

A mixture with other known active compounds, such as fungicides,insecticides, acaricides, nematicides, bird repellents, plant nutrientsand soil structure improvers is likewise possible.

Some of the safeners are already known as herbicides and accordingly, inaddition to the herbicidal action against harmful plants, also act byprotecting the crop plants.

The weight ratios of herbicide (mixture) to safener generally depend onthe herbicide application rate and the effectiveness of the safener inquestion and may vary within wide limits, for example in the range from200:1 to 1:200, preferably from 100:1 to 1:100, in particular from 20:1to 1:20. The safeners may be formulated analogously to the compounds ofthe formula (I) or their mixtures with other herbicides/pesticides andbe provided and used as a finished formulation or as a tank mix with theherbicides.

The required application rate of the compound of the formula (I) variesdepending, inter alia, on external conditions such as temperature,humidity and the type of herbicide used. It can vary within wide limits,for example between 0.001 and 10 000 g/ha or more of active substance;however, it is preferably between 0.5 and 5000 g/ha, particularlypreferably between 0.5 and 1000 g/ha and very particularly preferablybetween 0.5 and 500 g/ha.

When the transgenic plant of the invention contains one or more othergenes for tolerance towards other herbicides (as, for example, a genewhich encodes a mutated or unmutated EPSPS which confers on the planttolerance to glyphosate herbicides or a pat or bar gene conferringtolerance to glufosinate herbicides), or when the transgenic plant isnaturally resistant to another herbicide (such as sulfonylureatolerance), the method according to the invention can comprise thesimultaneous or chronologically staggered application of an HPPDinhibitor in combination with the said herbicide or herbicidecombination, for example glyphosate and/or glufosinate and/orsulfonylurea herbicides.

The invention also relates to the use of the chimeric gene encoding theHPPD of the invention as a marker gene during the transformation of aplant species, based on the selection on the abovementioned HPPDinhibitor herbicides.

The present invention also relates to a method for obtaining a plantresistant to a triketone or a pyrazolinate HPPD inhibitor, characterizedin that the plant is transformed with a chimeric gene expressing in theplant an HPPD of the invention as defined herein.

In a particular embodiment, the invention relates to said method forobtaining a plant resistant to a triketone or a pyrazolinate HPPDinhibitor, characterized in that the HPPD of the invention comprises SEQID No. 4 (from the amino acid position 2 to the amino acid position368), or a synthetic DNA encoding the HPPD of the invention adapted tothe codon usage of corn, rice, wheat, soya spp, sugarcane, onion,Brassica species plants, or cotton.

In another particular embodiment, the invention relates to said methodfor obtaining a plant resistant to a triketone HPPD inhibitor selectedfrom tembotrione, mesotrione, diketonitrile, isoxaflutole, sulcotrione,tefuryltrione, and bicyclopyrone.

In another particular embodiment, the invention relates to said methodfor obtaining a plant resistant to a triketone or a pyrazolinate HPPDinhibitor, characterized in that the plant also comprises aplant-expressible chimeric gene encoding a PDH (prephenatedehydrogenase) enzyme, or an enzyme with at least PDH.

The invention also relates to a method for controlling weeds in an areaor a field, which method comprises planting in this area or fieldtransformed plants resistant to a triketone or a pyrazolinate HPPDinhibitor which has been obtained according to the method describedabove, or transformed seeds which originates from them, and in applyinga dose which is toxic for the weeds of said triketone or pyrazolinateHPPD inhibitor without significantly affecting the said transformedseeds or the said transformed plants.

The invention also relates to a method for obtaining oil or mealcomprising growing a transformed plant resistant to a triketone or apyrazolinate HPPD inhibitor which has been obtained according to themethod described above, or a transformed seed which originates from suchplant, optionally treating such plant or seed with a triketone or apyrazolinate HPPD inhibitor, harvesting the grains and milling thegrains to make meal and extract the oil.

The invention also relates to the use of an HPPD of the invention asdescribed above, characterized in that the HPPD inhibitor is a triketoneHPPD inhibitor selected from tembotrione, mesotrione, topramezone,bicyclopyrone, tefuryltrione and sulcotrione.

The present invention also relates to a host organism, in particularplant cells or plants, which contain a chimeric gene comprising asequence encoding an HPPD according to the invention, and which alsocontain a gene functional in this host organism allowing overexpressionof a prephenate dehydrogenase (abbreviated herein as PDH) enzyme.

The term “PDH enzyme”, as used herein, refers to any natural or mutatedPDH enzyme exhibiting the PDH activity of conversion of prephenate toHPP. In particular, said PDH enzyme can originate from any type oforganism. An enzyme with PDH activity can be identified by any methodthat makes it possible either to measure the decrease in the amount ofprephenate substrate, or to measure the accumulation of a productderived from the enzymatic reaction, i.e. HPP or one of the cofactorsNADH or NADPH.

Many genes encoding PDH enzymes are described in the literature, andtheir sequences can be identified on the websitencbi.nlm.nih.gov/entrez. Particularly known is the gene encoding the PDHenzyme of the yeast Saccharomyces cerevisiae. (Accession No. S46037) asdescribed in Mannhaupt et al. (1989) Gene 85, 303-311, of a bacterium ofthe Bacillus genus, in particular of the species B. subtilis (AccessionNo. P20692) as described in Henner et al. (1986) Gene 49 (1) 147-152, ofa bacterium of the Escherichia genus, in particular of the species E.coli (Accession No. KMECTD) as described in Hudson et al. (1984) J. Mol.Biol. 180 (4), 1023-1051, or of a bacterium of the Erwinia genus, inparticular of the species E. herbicola (Accession No. S29934) asdescribed in Xia et al. (1992) J. Gen. Microbiol. 138 (7), 1309-1316.

The invention further relates to a method for obtaining a host organism,particularly a plant cell or a plant, resistant to an HPPD inhibitor byintegrating in such organism at least one nucleic acid sequence or onechimeric gene as defined above, and by further transforming it,simultaneously or successively, with a gene functional in this hostorganism allowing expression of a PDH (prephenate dehydrogenase) enzyme.In a particular embodiment, the invention relates to a method forobtaining a host organism, particularly a plant cell or a plant,resistant to a triketone or pyrazolinate HPPD inhibitor, particularlytembotrione, mesotrione topramezone, bicyclopyrone, isoxaflutole,pyrasulfotole, tefuryltrione, or sulcotrione.

Means and methods which could be used for obtaining a host organism,particularly a plant cell or a plant, transformed both with a geneallowing overexpression of an HPPD enzyme, and with a gene allowingoverexpression of a PDH enzyme are extensively described in WO04/024928, the content of which is hereby incorporated by reference.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgement or admission or any formof suggestion that such prior publication (or information) or knownmatter forms part of the common general knowledge in the field of thisinvention.

FIGURES

FIG. 1 Map of the plasmid pSE420::FMP29e

FIG. 2 Map of the T-DNA inserted into the tobacco plants

FIG. 3 Map of the T-DNA inserted in the different plants according toExamples 5 to 11; Abbreviations having the following meanings A, B, Cand G, tobacco plants, D, E and F, Zea mays plants, H, soybean plants,I, rice plants, and J, cotton plants. 35S: CaMV35S promoter, KanR: geneconferring resistance to the antibiotic kanamycin, nos: nopalinesynthase promoter, Ter: terminator, H6: sequence coding for an His TAG,OTP: optimized transit peptide, BAR (Bialaphos resistant, WO 8705629)and PAT (phosphinothricin N-Acetyltransferase, EP 257542):genesconferring tolerance to Bialaphos, phosphinothricin or glufosinate,2mEPSPS: gene coding for the double mutant (Thr102Ile and Pro106Ser)EPSPS (5-enolpyruvylshikimate synthase) from Zea mays (US 20030027312),2mAHAS: gene coding for the double mutant ALS (acetolactate synthase)from Arabidopsis (Pro197Ala and Trp574Leu; U.S. Pat. No. 5,378,824, HA:histone promoter from Arabidopsis gene, TEV: tobacco etch virus, FMP29e:gene coding for FMP29 optimized for the expression in E. coli with ansequence coding for an His TAG at its 5′ extremity, FMP29t: gene codingfor FMP29 optimized for the expression in dicotyledoneous plants with ansequence coding for an His TAG at its 5′ extremity, FMP29t-h, genecoding for FMP29 optimized for the expression in dicotyledoneous plants,FMP29m, gene coding for FMP29 optimized for the expression in Zea maysplants, LB, left border, RB, right border.

SEQUENCES LISTING

-   SEQ ID No. 1: Nucleic acid sequence encoding Picrophlus torridus    HPPD-   SEQ ID No. 2: Nucleic acid sequence encoding Picrophlus torridus    HPPD optimized for E. coli, plus containing at the 5′ end a nucleic    acid encoding an alanine and 6 histidine amino acids.-   SEQ ID No. 3: Nucleic acid sequence encoding Picrophlus torridus    HPPD optimized for Nicotiana tabaccum plus containing at the 5′ end    a nucleic acid sequence encoding an optimized transit peptide and an    HIS Tag.-   SEQ ID No. 4: Picrophlus torridus HPPD amino acid sequence derived    from SEQ ID No. 1-   SEQ ID No. 5: Protein encoded by SEQ ID No. 2-   SEQ ID No. 6: Picrophlus torridus HPPD amino acid sequence (SEQ ID    No. 4) fused with OTP (optimized transit peptide (WO 2009/144079))-   SEQ ID No. 7: Protein encoded by SEQ ID No. 3-   SEQ ID No. 8: Nucleic acid sequence encoding Arabidopsis thaliana    HPPD-   SEQ ID No. 9: Arabidopsis thaliana HPPD amino acid sequence-   SEQ ID No. 10: Protein encoded by SEQ ID No. 8 plus an additional    alanine directly downstream of the initial amino acid methionine    followed by 6 histidine amino acids-   SEQ ID No. 11: Protein of SEQ ID No. 9 plus the OTP sequence located    at the N-terminal extremity of the protein.-   SEQ ID No. 12: Protein of SEQ ID No. 10 plus the OTP sequence    directly located at the N-terminal extremity of the protein.-   SEQ ID No. 13: Primer sequence Xho-OTP-for-   SEQ ID No. 14: Primer sequence NcoI-OTP-rev-   SEQ ID No. 15: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for dicotyledoneous plants-   SEQ ID No. 16: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Zea mays plants-   SEQ ID No. 17: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Brassica napus plants-   SEQ ID No. 18: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Beta vulgaris plants-   SEQ ID No. 19: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Gossypium hirsitum plants-   SEQ ID No. 20: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Glycine max plants-   SEQ ID No. 21: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Hordeum vulgare plants-   SEQ ID No. 22: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Oryza sativa plants-   SEQ ID No. 23: Nucleic acid sequence encoding Picrophilus torridus    HPPD optimized for Triticum aestivum plants

EXAMPLES

The various aspects of the invention will be better understood with theaid of the experimental examples which follow. All the methods oroperations which are described below in these examples are given by wayof example and correspond to a choice which is made from among thedifferent methods which are available for arriving at the same orsimilar result. This choice has no effect on the quality of the resultand, as a consequence, any suitable method can be used by the skilledperson to arrive at the same or similar result. The majority of themethods for manipulating DNA fragments are described in “CurrentProtocols in Molecular Biology” Volumes 1 and 2, Ausubel F. M. et al.,published by Greene Publishing Associates and Wiley Interscience (1989)or in Molecular cloning, T. Maniatis, E. F. Fritsch, J. Sambrook, 1982,or in Sambrook J. and Russell D., 2001, Molecular Cloning: a laboratorymanual (Third edition)

Example 1

Preparation of Picrophlus torridus HPPD (named FMP29e) of SEQ ID No. 5and of the Arabidopsis thaliana HPPD identified by SEQ ID No. 10.

The Arabidopsis thaliana AtHPPD coding sequence (1335 bp; GenebankAF047834; WO 96/38567) was initially cloned into the expression vectorpQE-30 (QIAGEN, Hilden, Germany) in between the restriction sites ofBamHI and HindIII. The obtained vector was called “pQE30-AtHPPD”.

The original Picrophlus torridus HPPD sequence (1107 bp) coding for theprotein listed under the accession number Q6K798 at UniProtKB/TrEMBL wasmodified and synthesized using an Escherichia coli K12 optimized codonusage (Eurofins MWG operon (Ebersberg, Germany), GENEius software) andcloned in a modified pBluescript vector (Eurofins MWG operon, Ebersberg,Germany). In this vector, the sequence corresponding to the MCS(multiple cloning site) was partially removed that only the sequencescorresponding to the recognition of the restriction enzyme HindIIIremained on the both side of the insert.

At the 5′ end, directly downstream to the ATG was inserted a nucleicacid sequence coding for an alanine amino acid and a nucleic acidsequence encoding a N-terminal HIS6-Tag (6×HIS, encoded by: cat cac catcat cat cac). Upstream to the ATG, two additional cytosine base pairswere added in order to obtain a sequence corresponding to therecognition site of the restriction enzyme NcoI and downstream to thestop codon the sequences corresponding to the recognition site of therestriction enzyme XbaI were added. The resulting vector“pBluescript-FMP29e” was digested with the restriction enzymes NcoI andXbaI, the band migrating not to the length of the size of the vectorapproximately 3000 bp corresponding to the DNA was separated on anagarose gel per electrophoresis. Then the DNA coding for the HPPD waspurified using the MinElute™ Gel Extraction Kit (Qiagen, Hilden,Germany) and cloned into the pSE420(RI)NX vector (see below) previouslycut with the same restriction enzymes.

The cloning and expression vector pSE420(RI)NX (5261 bp) is based on theplasmid pSE420 by Invitrogen (Karlsruhe, Germany). Modifications of thisvector include the addition of a nptII gene (neomycinphosphotransferase; Sambrook and Russell, 2001, Molecular Cloning: alaboratory manual (Third edition)) conferring tolerance to theantibiotic kanamycin and is missing the majority of the superlinkerregion (multiple cloning site).

The plasmid possesses the trp-lac (trc) promoter and the lacI^(q) genethat provides the lac repressor in every E. coli host strain. The lacrepressor binds to the lac operator (lacO) and restricts expression ofthe target gene; this inhibition can be alleviated by induction withIsopropyl δ-D-1-thiogalactopyranoside (IPTG).

The resulting vector was called “pSE420(RI)NX-FMP29e” (see FIG. 1) andit was used to transform Escherichia coli BL21 cells (Merck, Darmstadt,Germany).

For the AtHPPD (Arabidopsis thaliana HPPD) that was used as referencesee WO 2009/144079.

Expression of HPPD was carried out in E. coli K-12 BL21 containingpQE30-AtHPPD or pSE420(RI)NX-FMP29e. Cells were allowed to grow until ODreached 0.5, then expression was initiated from the trp-lac (trc)promoter by induction with 1 mM IPTG which binds to the lac repressorand causes its dissociation from the lac operon. Expression was carriedout over 15 h at 28° C.

To prepare the pre-starter culture, 2 mL of TB medium (100 μg*mL⁻¹carbenicillin) were inoculated with 50 μL of an E. coli K-12 BL21glycerol stock. The pre-starter culture was incubated at 37° C. withshaking at 140 rpm for 15 h. 200 μl of the pre-starter culture was usedto initiate the starter culture (5 mL TB supplement with 100 μg*L⁻¹),which was incubated 3 h at 37° C.

To prepare the main culture, 400 mL of TB medium (100 μg*mL⁻¹carbenicillin) were inoculated with 4 mL of the starter culture. Thisstarter culture was incubated at 37° C. with shaking at 140 rpm untilOD₆₀₀ 0.5 was reached. Then recombinant protein expression was inducedwith 400 μl of 1M IPTG solution. The cells were allowed to grow for anadditional hour under these conditions, then the temperature was loweredto 28° C. and the culture was shaken at 140 rpm for 15 h. Cells wereharvested by centrifugation at 6000×g for 15 min at 4° C. Then cellpellets were stored at −80° C.

Isolation and Purification of His₆-AtHPPD and His₆-FMP29e in Native formLysis of Cells

Cells were lysed using Lysozyme, an enzyme that cleaves the1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamineresidues in peptidoglycan which forms the bacterial cell wall. Cellmembranes were then disrupted by the internal pressure of the bacterialcell. In addition, the lysis buffer contained Benzonase® Nuclease, anendonuclease that hydrolyzes all forms of DNA and RNA without damagingproteins and thereby largely reduces viscosity of the cell lysate. Lysisunder native conditions was carried out on ice.

For purification of His₆-tagged proteins the QIAexpress® Ni-NTA FastStart Kit was used following the user manual instruction.

Purification of His₆-Tagged Proteins by Immobilized Metal Ion AffinityChromatography (IMAC)

The cleared cell lysate (10 mL) obtained after centrifugation of thelysis reaction was loaded onto a Ni-NTA Fast Start Column from theQIAexpress® Ni-NTA Fast Start Kit (Qiagen, Hilden, Germany) andpurification was carried out according to the instruction manual. TheHis₆-tagged protein was eluted with 2.5 mL of elution buffer.

Desalting of HPPD Solutions by Gel Filtration

HPPD solutions eluted from a Ni-NTA Fast Start Column with 2.5 mL ofelution buffer were applied to a Sephadex G-25 PD-10 column (GEHealthcare, Freiburg, Germany) following the user manual instruction.After the whole sample had entered the gel bed, elution was performedwith 3.5 mL of storage buffer.

The HPPD solutions eluted from the desalting column were frozen at −80°C. in 1 mL aliquots.

Determination of HPPD protein concentration using the Bradford proteinassay Protein concentration was determined using the standard Bradfordassay (Bradford, (1976), Anal Biochem 72: 248-254).

Determination of Purity of HPPD Solutions Using SDS-PAGE

The integrity of the eluted protein was checked by SDS-PAGE protein gelelectrophoresis using the gel NuPAGE® Novex 4-12% Bis-Tris Gels(Invitrogen, Karlsruhe, Germany), approximately 10 μg of protein wereloaded. 10 μL of Laemmli Sample Buffer was added to 1-10 μL of proteinsolution and the mixture was incubated at 90° C. for 10 min. After shortcentrifugation step, the whole mixture was loaded into a slot of an SDSgel previously fixed in a XCell SureLock™ Novex Mini-Cell gel chamberfilled with NuPAGE® MOPS SDS Running Buffer (diluted from the20×-solution with ddH₂O). A voltage of 150 was then applied to the gelchamber for 1 h. For staining of protein bands, the gel was immersed inCoomassie Brilliant Blue R-250 Staining Solution. For destaining of thepolyacrylamide gel, it was immersed in Coomassie Brilliant Blue R-250Destaining Solution until protein bands appear blue on a white gel.

Example 2

Kinetic characterization and evaluation of tolerance to HPPD inhibitorsof HPPD enzymes “SEQ ID No. 5” and “SEQ ID No. 10”.

The HPPD activity was checked by the standard spectrophotmetric assay(method extensively described in WO 2009/144079)

Determination of HPPD In Vitro Kinetic Properties

K_(m), V_(max), and k_(cat) values for different HPPD enzymepreparations and K_(i), K₁=K_(on), and K₁=K_(off) for different HPPDinhibitors were determined using a HPLC assay for measurements of HPPDactivity. The assay mixtures contained in a volume of 1 ml 150 mMTris-HCI buffer at pH 7.8, 10 mM sodium ascorbate, 650 units of bovinecatalase (Sigma C30 (Sigma-Aldrich, Munich, Germany), 34 mg protein/ml,23,000 units/mg), and appropriate amounts of HPP, purified HPPD enzymeand HPPD inhibitors. For K_(m), V_(max), and k_(cat) value determinationHPP concentrations in the assay mixture were varied between 10 and 400μM. For K₁K₁=K_(on), and K₁=K_(off) value determination 2 mM HPP wasused. All assays were started by the addition of HPPD enzyme to theassay mixture and stopped at a series of times between 0 and 240 s byaddition of 200 μl of the reaction mixture to reaction assay tubescontaining 20 μl 10% perchloric acid. Precipitated protein was pelletedby a 5 minute centrifugation at 10,000 g. 100 μl of the supernatant wereloaded onto a 250×4 mm Knauer (Berlin, Germany) Eurospher 100-5C18-column equilibrated with 10% methanol, 0.1% trifluoroacetic acid(buffer A). The column was eluted, also at 1.5 ml/min, using a 4 minutewash with buffer A, followed by a 3 min wash with 95% methanol and by afurther 2 minute wash with buffer A. The elution of HGA (homogentisicacid) and HPP (hydroxyphenylpyruvate) was monitored at 292 nm. HGAelutes at around 5 minutes and HPP elutes later. A standard set ofconcentrations of HGA were used to provide a standard curve in order tocalibrate the 292 nm absorbance of the HGA peak versus HGAconcentration.

For K_(m) and V_(max) value determinations the initial rates of the HPPDreaction at different substrate concentrations were determined fromplots of HGA formed versus time and fitted to the Michaelis-Mentenequation for unireactant enzymes using the ID Business Solutions Ltd.(idbs.com) XLfit software suite. For the determination of K_(i),K_(I)=K_(on), and K⁻¹=K_(off) values the time-courses of the HPPDreaction at different inhibitor concentrations were fitted to theequations for Mechanism A, competitive inhibition, for tight-bindinginhibitors (Cha, S. (1975) Tight-binding inhibitors—I. Kineticbehaviour. Biochemical Pharmacology 24, 2177-2185) using the ID BusinessSolutions Ltd. XLfit software suite.

Determination of HPPD Activity in Presence of Several HPPD Inhibitors

In this content, pI₅₀-value means the log value of the concentration ofinhibitor necessary to inhibit 50% of the enzyme activity in molarconcentration.

pI₅₀-values for HPPD inhibitors were determined from dose-response plotsof HPPD activity versus inhibitor concentration using the assayextensively described in WO 2009/144079 at 2 mM fixed HPP concentrationand 3 minutes fixed incubation time using the ID Business Solutions Ltd.XLfit software suite.

TABLE 1 Determination of pI50 HPPD enzymes (Arabidopsis thaliana “SEQ IDNo. 10” and Picrophilus torridus “SEQ ID No. 5”) and their respectivetolerance to the several listed below HPPD inhibitors tembotrione,diketonitrile, mesotrione, bicyclopyrone, pyrasulfotole, sulcotrione,pyrazolate, tefuryltrione, and benzofenap. The symbol “>>” means thatthe value was far higher than the one indicated but could not beprecisely calculated within in the range of concentration of inhibitortested (2.5 × 10⁻⁶, 5.0 × 10⁻⁶, 1.0 × 10⁻⁵, 2.5 × 10⁻⁵, 6.3 × 10⁻⁵, and2.5 × 10⁻⁴M). Tembotrione Diketonitrile Mesotrione Bicyclopyrone SEQ IDNo. 10 >>5.6 >>5.6 >>5.6 5.2 SEQ ID No. 5 5.5 4.9 >>5.6 3.4Pyrasulfotole Sulcotrione Pyrazolate Tefuryltrione Benzofenap SEQ ID No.10 5.4 >>5.6 5.4 >>5.6 >>5.6 SEQ ID No. 5 4.0 >5.6 4.2 5.5. 4.8

TABLE 2 Determination of percentage of inhibition in presence of 5.0 ×10⁻⁶M inhibitors compared to the activity measured in absence of theinhibitor for the HPPD originated from Arabidopsis thaliana (SEQ ID No.10) and from Picrophilus torridus (SEQ ID No. 5). TembotrioneDiketonitrile Mesotrione Bicyclopyrone SEQ ID No. 10 92 87 86 29 SEQ IDNo. 5 58 26 84 24 Pyrasulfotole Sulcotrione Pyrazolate TefuryltrioneBenzofenap SEQ ID No. 10 69 74 61 100. 90 SEQ ID No. 5 7 92 16 87. 44

On the above Tables 1 and 2, it can be clearly seen, that theEuryarchaeota HPPD “SEQ ID No. 5” showed superior level of tolerance toall tested HPPD inhibitors than the plant at all tested HPPD inhibitorconcentrations than observed by employing the HPPD “SEQ ID No. 10” underidentical experimental conditions.

Example 3 Construction of Chimeric Genes for the Evaluation HPPDInhibitor Herbicide Tolerance in Tobacco Plants

A) Construction of the Chimeric Genes

The vector pRP-RD224 (extensively described in WO 2009/144079)containing the sequence coding for the OTP was used for PCR-mediatedattachment upstream of the nucleic acid sequence corresponding to therecognition site of the restriction enzyme XhoI and downstream of thenucleic acid sequence corresponding to the recognition site of therestriction enzyme NcoI. The obtained PCR product was cloned in thevector pCR®-Blunt II-TOPO® (Invitrogen, Karlsruhe, Germany) followingthe user manual instruction. The resulting vector was called“pCR-TOPO-OTP”. The insertion of the correct sequence was confirmed perstandard DNA sequencing. The DNA corresponding to the OTP was digestedwith the restriction enzymes NcoI and XhoI, separated per appropriategel electrophoresis and cloned into the plasmid pRT100 (Toepfer, (1987),Nucleic Acids Res 15:5890) previously and correspondingly digested withNcoI and XhoI restriction enzymes. The plasmid pRT100 is containing theCaMV35S promoter and CaMV35S terminator. The resulting vector wassubsequently digested with the restriction enzymes NcoI and XbaI. Thevector pSE420(RI)NX-FMP29e (see FIG. 1) was subjected to the restrictionenzymes NcoI and XbaI in order to obtain the DNA fragment correspondingto the “SEQ ID No. 2”. The resulting vector was digested by employingthe restriction enzyme HindIII to subclone theCaMV35S::OTP::FMP29e::CaMV35-term cassette (see FIG. 2) into the binaryvector pBin19 (Bevan (1984), Nucleic Acids Res. 12:8711-8721.)previously digested with the same enzyme and dephosphorylated. Theresulting vector was called “FMP29ebv”.

The vectors pQE-30-AtHPPD was used for PCR-mediated attachment of anNcoI restriction site and of a sequence encoding an N-terminal His₆-Tagto the 5′ ends and a XbaI restriction site to the 3′ ends of AtHPPD.

The PCR product of the AtHPPD gene was isolated from an agarose gel, cutwith the restriction enzymes NcoI and XbaI, purified with the MinElute™PCR Purification Kit (Qiagen, Hilden, Germany) and cloned into thepSE420(RI)NX vector cut with the same restriction enzymes.

The generated vector was called “pSE420(RI)NX-AtHPPD” and was digestedwith the restriction enzymes NcoI and XbaI and cloned into thepreviously opened vector pRT100 (Toepfer et al., (1987), Nucleic AcidsRes 15:5890) containing the CaMV35S promoter and CaMV35S terminator. Thegenerated vector was called “pRT100-AtHPPD”.

The vector pCR-TOPO-OTP was digested with the restriction enzymes NcoIand XhoI, and the DNA band corresponding to the OTP was cloned in thepreviously opened vector pRT100-AtHPPD with the above mentionedrestriction enzymes. The resulting vector was subsequently digested withrestriction enzyme HindIII and the expression cassette of interest wascloned into the previously opened and dephosphorylated binary vectorpBin19. The resulting vector was called “AtHPPDbv”.

The binary vectors FMP29ebv and AtHPPDbv were used to transformAgrobacterium tumefaciens (ATHV derived from EHA101) competent cellsselected on YEB media supplemented with the antibiotics kanamycin andrifampicin (extensively described in the patent applicationUS005925808A).

These Agrobacterium strains containing the binary vectors of interest(FMP29ebv or AtHPPDbv) were used to transform leaf discs from tobaccoNicotiana tabacum L. cv Samsun NN plants, having approximately a size of5×5 mm² as extensively described in Horsch et al., (1985), Science 227;1229-1231.

The leaf disks were co-cultivated for 2 days with Agrobacteriumtumefaciens cells containing either the binary vector FMP29ebv orAtHPPDbv. Then the leaf disks were transferred to a media allowing theregeneration of shoots for 6 weeks on MS (Musharige and Skoog, (1962),Physiol Plant 15 (3): 473-497) media supplemented with BAP (1 mg/mL;Benzylaminopurine), carbenicillin (250 mg/mL), cefotaxine (250 mg/mL),kanamycin (75 mg/mL) and tembotrione (10⁻⁶ M)

Regenerated calli were transferred on media to induce the development ofroots for 6 to 12 weeks: MS (1/2), supplemented with carbenicillin (250mg/mL), cefotaxine (250 mg/mL), kanamycin (75 mg/mL), and tembotrione(10⁻⁶ M).

After 6 weeks on this media, the shoots transformed with Agrobacteriumtumefaciens cells containing the binary vector AtHPPDbv, weretransferred on the same media depleted of HPPD inhibitor tembotrione.

The results are summarized on Table 5, below.

During the entire experiment, the plates containing the leaf disk werelocated in a growth chamber under controlled conditions (light 16 h,night 8 h, 25° C.).

Rooting of Calli

Regenerated shoot calli from a cell transformed with a nucleic acidsequence encoding an HPPD comprising SEQ ID No. 11 (Arabidopsisthaliana) or SEQ ID No. 7 (Picrophilus torridus) were transferred to amedia inducing root growth which media was further supplemented with theHPPD inhibitor tembotrione for 6 to 12 weeks. On none of the eventscontaining the HPPD defined by SEQ ID No. 11 (Arabidopsis thaliana) ornone transformed calli, root growth was observed under the above givenconditions. Contrary to this, under the identical conditions, the callicontaining the HPPD defined by SEQ ID No. 7 clearly developed numerousand healthy roots (see Table 3, below).

TABLE 3 Numbers of Events selected % Elongation & events rooted on formolecular rooting on 10⁻⁶M media without Calli containing: analysistembotrione tembotrione SEQ ID No. 11 21 0 5 SEQ ID No. 7 23 65 15

Leaf Disk Regeneration

Leaf disks were cut from plants containing HPPD SEQ ID No. 11(Arabidopsis thaliana) or SEQ ID No. 7 (Picrophilus torridus), followedby regeneration for 6 weeks under standard culture conditions on MSmedia supplemented with BAP (1 mg/mL; Benzylaminopurine), carbenicillin(250 mg/mL), cefotaxine (250 mg/mL) and further comprising one of thefollowing listed HPPD inhibitors at the mentioned concentration(tembotrione (10⁻⁶ M), diketonitrile (5·10⁻⁶ M), Mesotrione (10⁻⁶ M) andbicyclopyrone (10⁻⁶ M)) with a media containing none HPPD inhibitors asthe positive control. At the end of the experiments the level ofregeneration was evaluated as followed:

“−” means that the leaf disks looked the same as leaf disk from wildtype tobacco plants on media supplemented with the inhibitor mentionedabove.

“++++” means that the leaf disks looked like the leaf disks from thewild type tobacco plants on media without inhibitor.

“+”, “++”, and “+++” indicate regenerated leaf disks were heavily (+),medium (++) and less (+++) affected by the presence of the inhibitors.

The results of the experiments are summarized in Table 4.

TABLE 4 Effects of various HPPD inhibitors the regeneration of leaf diskoriginating from transgenic plants comprising either a gene coding foran HPPD obtained from Arabidopsis (SEQ ID No. 11) or from Picrophilustorridus SEQ ID No. 7. Leaf disks containing Control TembotrioneDiketonitrinile Mesotrione Bicyclopyrone SEQ ID NO 11 ++++ − − − − SEQID NO 7 ++++ +++ ++ ++ ++

Whereas in case of plants containing HPPD defined by SEQ ID No. 7(Picrophilus torridus) showed the same or only slightly reducedregeneration compared to this un-treated control, the correspondingplants containing HPPD defined by SEQ ID No. 11 (Arabidopsis thaliana)did not show any regeneration but developed clearly visible bleachingphenotype compared to the untreated control in the presence of alltested HPPD inhibitors.

Example 4 Glasshouse Trials to Evaluate Tolerance to HPPD InhibitorHerbicides of Transgenic Tobacco Plants Expressing a Gene Coding forTolerant HPPD Protein

Preparation of transgenic plant lines expressing either Arabidopsis orFMP29 HPPD enzymes. Glasshouse testing for herbicide tolerance.

Response to tembotrione, isoxaflutole and bicyclopyrone

T0 Tobacco plants containing either the gene from Arabidopsis coding forHPPD or the gene FMP29e from Picrophilus torridus coding for FMP29 HPPD,mentioned above (Example 3), were transferred to the glasshouse (28/20°C.), to develop further and produce seeds. Those seeds were harvestedand put on soil (ED73 mixed with sand and osmocote Pro) to germinate inthe glasshouse (28/20° C.). Three to four weeks later, plantlets weretransferred to single pots containing the soil mentioned above. Twoweeks later, plants of a size 4-6 cm diameter were sprayed with either

-   -   tembotrione at 100 gAl/ha prepared from a WP20 (wettable powder        20%) formulation supplemented with ammonium sulfate and methyl        ester raps oil, or    -   isoxaflutole at 100 gAl/ha prepared from a WP20 formulation        supplemented with ammonium sulfate and methyl ester raps oil, or    -   bicyclopyrone at 100 gAl/ha prepared from a WP20 formulation        supplemented with ammonium sulfate and methyl ester raps oil, or    -   “blind formulation” made from a WP20 formulation without active        ingredient (Al) supplemented with ammonium sulfate and methyl        ester raps oil, and were then transferred to a growth chamber        with adequate light conditions (20000 Lux).

Seven days after the application (DAT) of the different herbicides, thesymptoms in tranformed plants were evaluated in comparison to theresponse observed on the wild type tobacco plants sprayed at the sametime and under the same conditions as the tobacco plants containing thetransgenes (100% means the plants displayed the same bleaching phenotypeas the wild type plants, 0% means that the plants looked like the wildtype plants treated with the “blind formulation”, and intermediatepercentage represent the degree of observed symptoms).

TABLE 5 Wild type tobacco plants (A) and T1 populations of tobaccoevents containing alternatively, the expression cassettes describedabove having the promoter CaMV 35S, the sequence coding for OTP and thesequence coding for Arabidopsis HPPD (B) or the promoter CaMV35S, thesequence encoding OTP, and the sequence FMP29e coding for the HPPD FMP29(C). Assessments of herbicidal damage at 7 days after application (DAT)per spray with 100 g Al/ha of tembotrione or isoxaflutole supplementedwith ammonium sulfate and methyl ester raps oil. It is clear that plantscontaining FMP29e gene were far more tolerant to tembotrione andisoxaflutole. Plants belonging to categories (B) and (C) have not beenselected for the presence of the respective transgene prior to theherbicide application. % injury, 7 DAT, 100 g Al/ha Line TembotrioneIsoxaflutole A WT 1 100 100 Wild Type WT 2 100 100 WT 3 100 100 WT 4 10098 WT 5 100 99 WT 6 100 99 WT 7 100 100 WT 8 100 n.d. WT 9 100 n.d. WT10 100 n.d. WT 11 100 n.d. WT 12 100 n.d. WT 13 100 n.d. WT 14 100 n.d.B 258 1 100 100 Arabidopsis HPPD 258 2 100 100 258 3 100 100 258 4 100100 258 5 100 100 258 6 30 100 252 1 30 30 252 2 40 70 252 3 40 95 252 440 98 252 5 50 98 252 6 60 99 252 7 60 99 252 8 70 99 252 9 70 99 252 1275 100 252 13 75 100 252 14 75 100 252 15 80 100 327 1 10 10 327 2 20 20327 3 20 60 327 4 40 60 327 5 50 70 327 6 50 80 327 7 70 95 327 8 70 98327 9 70 99 327 10 70 100 327 11 70 100 327 12 80 100 327 13 80 100 32714 80 100 327 15 80 100 C 115 1 30 0 FMP29e 115 2 40 2 115 3 50 5 115 450 10 115 5 50 15 115 6 n.d. 30 115 7 n.d. 30 115 8 n.d. 40 292 1 20 20292 2 20 30 292 3 30 40 292 4 40 n.d. 292 5 40 n.d.

Response to Bicyclopyrone.

Seeds of wild type tobacco plants and T1 tobacco plants carrying thegene from Picrophilus torridus FMP29e coding for HPPD were sown on MSmedia (Murashige and Skoog 1964) supplemented with 50 g/L kanamycin.After 4 weeks, rooted green plantlets were transferred to soil and grownfor 3 weeks in the glasshouse as described above then sprayed with amixture containing bicyclopyrone (100 g Al/ha), ammonium sulfate andmethyl ester raps oil. The plants were classified in two categoriesbased on the phenotype developed in response to the herbicide seven daysafter the treatment. Class I was defined as plants displayed no injuriesto light injuries in response to the herbicide treatment (injury:0-30%), Class II was defined as plants displaying strong injuries tosimilar injuries as seen with wild type plants submitted to the sametreatment (injury: 31-100%). In this case, only plants containing atleast one T-DNA were exposed to the herbicidal treatment. In general, itcan be seen that even the plants containing only one T-DNA insertalready showed up a significant and sufficient level of tolerance to anexpose a field dose of the HPPD inhibitor herbicide bicyclopyrone.

TABLE 6 Bicyclopyrone, 100 g Al/ha 7 DAT % of tolerant Transgene LineClass I Class II plant — WT 0 12 0 FMP29e 121 38 11 2

The plants containing the HPPD FMP29 displayed tolerance to the HPPDinhibitor herbicide bicyclopyrone.

It can be summarized from the above presented data, that the plantsexpressing the gene FMP29e from Picrophilus torridus coding for theFMP29 HPPD obtained from several independent transgenic events arehighly tolerant to several HPPD inhibitor herbicides at doses appliedunder standard agronomic conditions.

Example 5 Construction of Binary Vectors to Express SeveralDicotyledoneous Optimized Variants in Plants and Glasshouse Trial toEvaluate Tolerance of Tobacco Plants Containing Such Variants

Cloning into pBin19 of FMP29t (SEQ ID No. 3), FMP29t-h (SEQ ID No. 15)

A gene with codon usage optimized for the expression in dicotyledoneousplants coding for the HPPD protein FMP29 were designed, and namedFMP29t-h (SEQ ID No. 15) and the same gene with an additional sequencescoding for an OTP and for an HIS TAG at its 5′ extremity called FMP29t(SEQ ID No. 3). The sequence corresponding to FMP29t-h gene was clonedusing the restriction enzymes NcoI and XbaI in the previously describedvector pRT100-OTP, containing a CaMV35S promoter and terminator. Theresulting vector was called pRT100-OTP-FMP29t-h. The sequencecorresponding to FMP29t was cloned in the previously described vectorpRT100 using the restriction enzymes XhoI and XbaI, and the resultingvector was called pRT100-OTP-FMP29t. The fragments corresponding toPromCaMV35S-OTP-FMP29t-h-TerCaMV35S andPromCaMV35S-OTP-HIS6-FMP29t-TerCaMV35S were subclone in the pBIN19vector (described above) using the restriction enzyme Sbfl. The binaryvectors were respectively called pBin19-FMP29t-h (FIG. 3C) andpBin19-FMP29t (FIG. 3B) and can be used for example to transformdicotyledenous plants, such as tobacco plants as described above.Sufficiently grown transformant plants are then tested for theirtolerance to HPPD inhibitor herbicides, such as tembotrione. Thedevelopment of the observed symptoms in response to the herbicidaltreatment is evaluated and compared to the response of wild type plantsunder the same conditions.

Example 6 Cloning of Gene FMP29e, FMP29t and FMP29m Coding for FMP29HPPD in a Vector to Transform Zea mays Plants

FMP29e (SEQ ID No. 2), FMP29t (SEQ ID No. 3), FMP29m-h (SEQ ID No. 16)

a—FMP29e in pHoe6/Ac: Gene with a codon usage optimized for E. coli,plus at its 5′ extremity a sequence coding for OTP and sequence codingfor an His TAG.

The vector pRT100-FMP29e containing the gene coding for the HPPD FMP29,optimized for the expression in E. coli under the control of the CaMV35Spromoter, was digested with the restriction enzyme HindIII. The

CaMV35S::OTP::FMP29e::CaMV35S-term cassette was further cloned into thebinary vector pHoe6/Ac (U.S. Pat. No. 6,316,694) previously digestedwith the same restriction enzyme and dephosphorylated. The resultingvector was called pHoe6/Ac/FMP29e.

b—FMP29t in pHoe6/Ac (SEQ ID No. 3): Gene with a codon usage optimzedfor dicotyledoneous plants, plus at its 5′ extremity a sequence codingfor OTP and sequence coding for an His TAG.

FMP29t in pRT100. A version of the gene coding for the protein FMP29optimized for the expression in Nicotiana tobaccum plus containing atthe 5′ end a nucleic acid sequence encoding an optimized transit peptideand an HIS tag was ordered and called FMP29t. Upstream to this sequencewas added the recognition sequence for the restriction enzyme XhoI anddownstream the recognition sequence for the restriction enzyme XbaI. TheDNA corresponding to the OTP and FMP29t were digested with therestriction enzymes XhoI and XbaI, separated per appropriate gelelectrophoresis and cloned into the vector pRT100 (Toepfer, (1987),Nucleic Acid Res 15:5890) previously digested with XhoI and NcoIrestriction enzymes. The plasmid pRT100 contains the CaMV35S promoterand CaMV35S terminator. The resulting vector was called pRT100-FMP29t,and digested with the restriction enzyme HindIII to separate the DNAcorresponding to CaMV35S::OTP::FMP29t::CaMV35S-term cassette from therest of the vector, in order to clone it into the previously restrictedvector pHoe6/Ac (U.S. Pat. No. 6,316,694). The resulting vector wascalled pHoe6/Ac/FMP29t (FIG. 3).

c—FMP29m in pHoe6/Ac (SEQ ID No. 16): Gene with a codon usage optimzedfor monocotyledoneous plants plus at its 5′ extremity a sequence codingfor OTP. FMP29m in pRT100-OTP (NcoI-XbaI) then HindIII

The variant of the gene optimized for the expression in monocotyledonplants coding for FMP29, called FMP29m was ordered, and upstream of thestart codon was added a NcoI restriction site while downstream of thestop codon was added the recognition sequence for the restriction enzymeXbaI. The DNA sequence corresponding to FMP29m was digested with therestriction enzymes NcoI and XbaI, then separated per gelelectrophoresis, finally isolated from the gel. The isolated DNAfragment was mixed with the vector pRT100-OTP (mentioned above)previously also digested with the same restriction enzymes. Theresulting vector was called pRT100-OTP-FMP29m, containing the expressioncassette CaMV35S::OTP::FMP29m::CaMV35Sterm, which was isolated using therestriction enzyme HindIII then further cloned into the previouslyopened and dephosphorylated vector pHOE6/Ac containing the gene codingfor the PAT (Phosphinothricin acetyl transferase) enzyme, conferringresistance to the herbicide glufosinate (U.S. Pat. No. 6,316,694). Theresulting plasmid was called pHoe/Ac/FMP29m (FIG. 3F)

Maize Transformation:

The plasmids pHoe6/Ac (U.S. Pat. No. 6,316,694), pHoe6/Ac/FMP29e,pHoe6/Ac/FMP29t and pHoe6/Ac/FMP29m were used to transform maizeculture.

The maize culture, protoplast isolation, transformation and regenerationof fertile transgenic maize plants have been performed according to theU.S. Pat. No. 6,284,945, “Zea mays (L.) with capability of long term,highly efficient plant regeneration including fertile transgenic maizehaving a heterologous gene, and their preparation”. Transformed calliwere selected on media containing phosphinothricin. Regenerated rootedplants were then transferred to soil, and allowed to grow and produceseeds in the glasshouse under standard conditions (28/20° C.). Adultplants were grown until seed production and seeds were collected forfurther sowing, sufficiently developed plants will be treated with therespective HPPD inhibitor herbicides.

Example 7 Construction of Binary Soybean Transformation Vectors

A binary vector for soybean transformation is, for example, constructedwith the CaMV35 promoter driving the expression of the gene FMP29t-h(SEQ ID No. 15), with a codon usage optimized for the expression indicotyledoneous plants and at its 5′ extremity was added a sequencecoding for an OTP, and further upstream a sequence TEV (Tobacco etchvirus) to improve the stability of the mRNA in plants followed by theCaMV35S terminator. The nucleotide sequence of the gene FMP27t-h isgiven in SEQ ID No. 15. Additionally, the transformation vector alsocontains a PAT gene cassette in which the gene is driven by a CaVM35Spromoter and followed by a CaMV35S terminator for glufosinate basedselection during the transformation process and a 2mEPSPS gene cassettein which the gene is driven by an histone promoter from Arabidopsis toconfer tolerance to the herbicide glyphosate to the transformed plants(see FIG. 3H). The binary vector was called pFCO115.

Example 8 Soybean T0 Plant Establishment and Selection

Soybean transformation is achieved using methods well known in the art,such as the one described using the Agrobacterium tumefaciens mediatedtransformation soybean half-seed explants described by Paz et al. (2006,Plant cell Rep. 25:206). Transformants were identified usingIsoxaflutole as selection marker. The appearance of green shoots wasobserved, and documented as an indicator of tolerance to the herbicideisoxaflutole.

Tolerant green shoots were transferred to rooting media or grafted.Rooted plantlets were transferred to the glasshouse after an acclimationperiod.

Plants containing the transgene are then sprayed with HPPD inhibitorherbicides, as for example with tembotrione at a rate of 100 g Al/ha.Ten days after the application the symptoms due to the application ofthe herbicide will be evaluated and compared to the symptoms observed ona wild type plants under the same conditions.

Example 9 Construction of Binary Cotton Transformation Vectors

A binary vector for cotton transformation is, for example, constructedwith the CaMV35 promoter driving the expression of the gene FMP29t-h(SEQ ID No. 15), with a codon usage optimized for the expression indicotyledoneous plants and at its 5′ extremity was added a sequencecoding for an OTP, and further upstream a sequence TEV (Tobacco etchvirus) to improve the stability of the mRNA in plants followed by theCaMV35S terminator. The nucleotide sequence of the gene FMP27t-h isgiven in SEQ ID No. 15. Additionally, the transformation vector alsocontains a PAT gene cassette in which the gene is driven by a CaVM35Spromoter and followed by a CaMV35S terminator for glufosinate basedselection during the transformation process and a 2mEPSPS gene cassettein which the gene is driven by an histone promoter from Arabidopsis toconfer tolerance to the herbicide glyphosate to the transformed plants(see FIG. 3J).

Example 10 Cotton T0 Plant Establishment and Selection

Cotton transformation is achieved using methods well known in the art,especially preferred method in the one described in the PCT patent WO00/71733.

Regenerated plants are transferred to the glasshouse. Following anacclimation period, sufficiently grown plants are sprayed with HPPDinhibitor herbicides as for example tembotrione 100 gAl/ha supplementedwith ammonium sulfate and methyl ester raps oil. Seven days after thespray application, the symptoms due to the treatment with the herbicideare evaluated and compared to the symptoms observed on wild type cottonplants subjected to the same treatment under the same conditions.

Example 11 Construction of Binary Transformation Vectors to GeneratePlants Tolerants to Four Herbicides with Distinct Modes of Action

A binary vector for dicotyledoneous plant transformation is, forexample, constructed with the CaMV35 promoter driving the expression ofthe gene FMP29t-h (SEQ ID No. 15), with a codon usage optimized for theexpression in dicotyledoneous plants and at its 5′-extremity was added asequence coding for an OTP followed by the CaMV35S terminator. Thenucleotide sequence of the gene FMP29t-h is given in SEQ ID No. 15.Additionally, the transformation vector also contains a PAT genecassette in which the gene is driven by a CaVM35S promoter and followedby a CaMV35S terminator to confer tolerance to glufosinate to the plantexpressing the gene, a 2mEPSPS gene cassette coding for the doublemutant (Thr102Ile and Pro106Ser) EPSPS in which the gene is driven by anhistone promoter from Arabidopsis to confer tolerance to the herbicideglyphosate to the transformed plants, and an Arabidopsis thaliana 2mAHASgene cassette encoding a tolerant ALS enzyme (Acetolactate, Pro197Ala,Trp574Leu) driven by a CaMV35S promoter to confer tolerance toherbicides from the sulfonylurea or imidazolinone classes to the plantexpressing this gene (see FIG. 3G).

The gene cassettes is finally cloned into the vector pHoe6/Ac (U.S. Pat.No. 6,316,694), and the final vector is calledpHoe6/FMP29t-h/PAT/EPSPS/AHAS, and is used to transform dicotyledoneousplants via Agrobacterium tumefaciens mediated state of the art methods.T0 plants are transferred to soil, and after an acclimation period,sufficiently grown plants are sprayed successively with an herbicidefrom the HPPD inhibitor class, then with glyphosate, then withglufosinate and finally with an herbicide from the sulfonylurea classfor example.

Example 12 Generation of Transgenic Plants Showing Tolerance toHerbicides of Three Different Mode of Action

A binary vector for tobacco transformation is, for example, constructedwith the CaMV35 promoter driving the expression of the gene FMP29t-h(SEQ ID No. 15), with a codon usage optimized for the expression indicotyledoneous plants and at its 5′ extremity was added a sequencecoding for an OTP, and further upstream a sequence TEV (Tobacco etchvirus) to improve the stability of the mRNA in plants followed by theCaMV35S terminator. The nucleotide sequence of the gene FMP29t-h isgiven in SEQ ID No. 15. Additionally, the transformation vector alsocontains a PAT gene cassette in which the gene is driven by a CaVM35Spromoter and followed by a CaMV35S terminator for glufosinate basedselection during the transformation process and a 2mEPSPS gene cassettein which the gene is driven by an histone promoter from Arabidopsis toconfer tolerance to the herbicide glyphosate to the transformed plants(see FIG. 3H). The binary vector was called pFCO115. The above vectorwas used to transform leaf dics obained from Nicotiana tobacum plants,according to Example 3.

Transgenic tobacco plants were transferred to the greenhouse and treatedwith glyphosate at a rate of 1121 g Al/ha. Seeds were produced from suchtolerant tobacco plants and harvested. These seeds were put on soil togerminate in the glasshouse. Three to four weeks later, 50 plantlets perevent were transferred to single pots. Two weeks later, plants of a size4-6 cm are sprayed either with:

glufosinate-ammonium 1000 g Al/ha, glyphosate 1121 g Al/ha, tembotrione 100 g Al/ha, or tembotrione + glyphosate  100 g Al/ha + 1121 g Al/ha

After nine days, the symptoms caused by the respective heribciceapplications are evaluated

The invention claimed is:
 1. A method for controlling weeds in an areaor a field which contains a plant or a seed, which method comprisesapplying, to the area or the field, a dose of a HPPD inhibitor herbicidewhich is toxic for said weeds, without significantly affecting the seedor plant, wherein the plant or the seed comprises a chimeric genecomprising a coding sequence operably linked to a plant-expressiblepromoter, the coding sequence comprises a nucleic acid sequence whichencodes a Picrophilus torridus hydroxyphenylpyruvate dioxygenase (HPPD)protein according to SEQ ID No. 4 from amino acid position 2 to aminoacid position
 368. 2. The method for controlling weeds according toclaim 1, characterized in that the HPPD inhibitor herbicide is selectedfrom the group consisting of isoxaflutole, tembotrione, mesotrione,sulcotrione, pyrasulfotole, topramezone,2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-CF₃-phenyl)propane-1,3-dione and2-cyano-3-cyclopropyl-1-(2-SO₂CH₃-4-2,3 Cl₂ phenyl)propane-1,3-dione,bicyclopyrone, benzobicyclon, tefuryltrione, diketonitrile, andpyrazoxyfen.
 3. A method for obtaining oil or meal comprising growing aplant comprising a chimeric gene comprising a coding sequence operablylinked to a plant-expressible promoter, the coding sequence comprises anucleic acid sequence which encodes a Picrophilus torridushydroxyphenylpyruvate dioxygenase (HPPD) protein according to SEQ ID No.4 from amino acid position 2 to amino acid position 368, optionallytreating the plant with a HPPD inhibitor herbicide, harvesting grainsfrom the plant and milling the grains to make meal, and optionallyextracting oil from the grains.
 4. The method of claim 1, wherein themethod is for controlling weeds in an area or a field which containssaid plant.
 5. The method of claim 1, wherein the method is forcontrolling weeds in an area or a field which contains said seed.
 6. Themethod of claim 1, wherein the chimeric gene comprises the nucleotidesequence of SEQ ID No. 1 from nucleotide position 4 to nucleotideposition 1107, or SEQ ID No. 3 from nucleotide position 396 tonucleotide position
 1503. 7. The method of claim 1, wherein the chimericgene comprises, upstream of the HPPD coding sequence, a nucleic acidsequence which encodes a transit peptide active in plants so that atransit peptide/HPPD fusion protein is encoded by said chimeric gene. 8.The method of claim 1, wherein the plant or the seed further comprises achimeric gene encoding a prephenate dehydrogenase (PDH) enzyme.
 9. Themethod of claim 1, wherein the plant or the seed further comprises oneor more chimeric genes conferring tolerance to (a) a growth regulatorherbicide; or (b) a herbicide inhibiting enzyme, wherein the enzyme isselected from the group consisting of (i) acetolactate synthase, (ii)5-enolpyruvylshikimate (EPSP) synthase and (iii) glutamine synthase; or(c) a combination thereof.
 10. The method of claim 9, wherein the growthregulator herbicide is selected from the group consisting of 2,4-D anddicamba.
 11. The method of claim 3, wherein the plant is selected fromthe group consisting of soya, corn, cotton, and grain.